U.S. patent application number 14/732603 was filed with the patent office on 2016-01-14 for novel poly(adp-ribose) polymerase genes.
The applicant listed for this patent is ABBVIE DEUTSCHLAND GMBH & CO. KG. Invention is credited to Thomas HoGER, Michael Kock, Burkhard Kroger, Hans-Georg Lemaire, Wilfried Lubisch, Bernd Otterbach.
Application Number | 20160010067 14/732603 |
Document ID | / |
Family ID | 26046644 |
Filed Date | 2016-01-14 |
United States Patent
Application |
20160010067 |
Kind Code |
A1 |
Kock; Michael ; et
al. |
January 14, 2016 |
NOVEL POLY(ADP-RIBOSE) POLYMERASE GENES
Abstract
The invention relates to poly(ADP-ribose)polymerase (PARP)
homologs which have an amino acid sequence which has a) a
functional NAD.sup.+ binding domain and b) no zinc finger sequence
motif of the general formula TABLE-US-00001
CX.sub.2CX.sub.mHX.sub.2C in which m is an integral value from 28
or 30, and the X radicals are, independently of one another, any
amino acid; and the functional equivalents thereof; nucleic acids
coding therefor; antibodies with specificity for the novel protein;
pharmaceutical and gene therapy compositions which comprise
products according to the invention; methods for the analytical
determination of the proteins and nucleic acids according to the
invention; methods for identifying effectors or binding partners of
the proteins according to the invention; novel PARP effectors; and
methods for determining the activity of such effectors.
Inventors: |
Kock; Michael;
(Schifferstadt, DE) ; HoGER; Thomas;
(Edingen-Neckarhausen, DE) ; Kroger; Burkhard;
(Limburgerhof, DE) ; Otterbach; Bernd;
(Ludwigshafen, DE) ; Lubisch; Wilfried;
(Heidelberg, DE) ; Lemaire; Hans-Georg;
(Limburgerhof, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ABBVIE DEUTSCHLAND GMBH & CO. KG |
WIESBADEN |
|
DE |
|
|
Family ID: |
26046644 |
Appl. No.: |
14/732603 |
Filed: |
June 5, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12778692 |
May 12, 2010 |
9051553 |
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14732603 |
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09701586 |
Nov 30, 2000 |
7754459 |
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PCT/EP99/03889 |
Jun 4, 1999 |
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12778692 |
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Current U.S.
Class: |
424/139.1 ;
424/94.5; 435/15; 435/193; 435/252.31; 435/252.33; 435/252.34;
435/252.35; 435/254.21; 435/254.3; 435/320.1; 435/348; 435/358;
435/419; 435/6.11; 435/6.12; 435/7.4; 514/44R; 530/387.9; 536/23.2;
800/16; 800/18 |
Current CPC
Class: |
C12Q 1/48 20130101; C12N
9/1077 20130101; C07K 2317/76 20130101; C07K 16/40 20130101; A01K
2217/05 20130101; A61K 49/00 20130101; A61P 43/00 20180101; A61K
38/00 20130101; C07K 2319/00 20130101; C12Y 204/0203 20130101; G01N
2333/91142 20130101 |
International
Class: |
C12N 9/10 20060101
C12N009/10; C12Q 1/48 20060101 C12Q001/48; C07K 16/40 20060101
C07K016/40 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 5, 1998 |
DE |
19825213.7 |
Mar 1, 1999 |
DE |
19908837.3 |
Claims
1. A poly(ADP-ribose) polymerase (PARP) homolog derived from a
human or non-human mammal which has an amino acid sequence which
has a) a functional NAD.sup.+ binding domain and b) no zinc finger
sequence motif of the general formula TABLE-US-00011
CX.sub.2CX.sub.mHX.sub.2C
in which m is an integral value from 28 or 30, and the X radicals
are, independently of one another, any amino acid.
2. A PARP homolog as claimed in claim 1, wherein the functional
NAD.sup.+ binding domain comprises one of the following general
sequence motifs: TABLE-US-00012 PX.sub.n(S/T)GX.sub.3GKGIYFA,
(S/T)XGLR(I/V)XPX.sub.n(S/T)GX.sub.3GKGIYFA or
LLWHG(S/T)X.sub.7IL(S/T)XGLR(I/V)XPX.sub.n(S/T)GX.sub.3GKGIYFAX.sub.3SKS
AXY
in which n is an integral value from 1 to 5, and the X radicals
are, independently of one another, any amino acid.
3. A PARP homolog as claimed in claim 1, comprising at least
another one of the following part-sequence motifs: TABLE-US-00013
LX.sub.9NX.sub.2YX.sub.2QLLX (D/E)X.sub.10/11WGRVG,
AX.sub.3FXKX.sub.4KTXNXWX.sub.5FX.sub.3PXK,
QXL(I/L)X.sub.2IX.sub.9MX.sub.10PLGKLX.sub.3QIX.sub.6L,
FYTXIPHXFGX.sub.3PP; and KX.sub.3LX.sub.2LXDIEXAX.sub.2L,
in which the X radicals are, independently of one another, any
amino acid.
4. A PARP homolog as claimed in claim 1, selected from human PARP
homologs, which has the amino acid sequence shown in SEQ ID NO: 2
(human PARP2) or SEQ ID NO: 4 or 6 (human PARP3 type 1 or 2); or
murine PARP homologs which have the amino acid sequence shown in
SEQ ID NO:8 (mouse PARP long form) or SEQ ID No:10 (mouse PARP
short form).
5. A binding partner for having specificity for PARP homologs as
claimed in claim 1, selected from a) antibodies and fragments
thereof, b) protein-like compounds which interact with a part
sequence of the protein, and c) low molecular weight effectors
which modulate the catalytic PARP activity or another biological
function of a PARP molecule.
6. A nucleic acid comprising a) a nucleotide sequence coding for at
least one PARP homolog as claimed in claim 1, or the complementary
nucleotide sequence thereof; b) a nucleotide sequence which
hybridizes with a sequence as specified in a) under stringent
conditions; or c) nucleotide sequences which are derived from the
nucleotide sequences defined in a) and b) through the degeneracy of
the genetic code.
7. A nucleic acid as claimed in claim 6, comprising a) nucleotides
+3 to +1715 shown in SEQ ID NO:1; b) nucleotides +242 to +1843
shown in SEQ ID NO:3; c) nucleotides +221 to +1843 shown in SEQ ID
NO:5; d) nucleotides +112 to +1710 shown in SEQ ID NO:7; or e)
nucleotides +1 to +1584 shown in SEQ ID NO:9.
8. An expression cassette comprising, under the genetic control of
at least one regulatory nucleotide sequence, at least one
nucleotide sequence as claimed in claim 6.
9. A recombinant vector comprising at least one expression cassette
as claimed in claim 8.
10. A recombinant microorganism comprising at least one recombinant
vector as claimed in claim 9.
11. A transgenic mammal comprising a vector as claimed in claim
9.
12. A PARP-deficient mammal or PARP-deficient eukaryotic cell, in
which functional expression of at least one gene which codes for a
PARP homolog as claimed in claim 1 is inhibited.
13. An in vitro detection method for PARP inhibitors, which
comprises a) incubating an unsupported or supported polyADP
ribosylatable target with a reaction mixture comprising a1) a PARP
homolog as claimed in claim 1, a2) a PARP activator; and a3) a PARP
inhibitor or an analyte in which at least one PARP inhibitor is
suspected; b) carrying out the polyADP ribosylation reaction; and
c) determining the polyADP ribosylation of the target qualitatively
or quantitatively.
14. The method as claimed in claim 13, wherein the PARP homolog is
preincubated with the PARP activator and the PARP inhibitor or an
analyte in which at least one PARP inhibitor is suspected, before
the polyADP ribosylation reaction is carried out.
15. The method as claimed in claim 13, wherein the
polyADP-ribosylatable target is a histone protein.
16. The method as claimed in claim 13, wherein the PARP activator
is activated DNA.
17. The method as claimed in claim 13, wherein the polyADP
ribosylation reaction is started by adding NAD.sup.+.
18. The method as claimed in claim 13, wherein the polyADP
ribosylation of the supported target is determined using
anti-poly(ADP-ribose) antibodies.
19. The method as claimed in claim 13, wherein the unsupported
target is labeled with an acceptor fluorophore.
20. The method as claimed in claim 19, wherein the polyADP
ribosylation of the unsupported target is determined using
anti-poly(ADP-ribose) antibody which is labeled with a donor
fluorophore which is able to transfer energy to the acceptor
fluorophore.
21. The method as claimed in claim 19, wherein the target is
biotinylated histone, and the acceptor fluorophore is coupled
thereto via avidin or streptavidin.
22. The method as claimed in claim 20, wherein the
anti-poly(ADP-ribose) antibody carries a europium cryptate as donor
fluorophore.
23. An in vitro screening method for binding partners for a PARP
molecule, which comprises a1) immobilizing at least one PARP
homolog as claimed in claim 1 on a support; b1) contacting the
immobilized PARP homolog with an analyte in which at least one
binding partner is suspected; and c1) determining, where
appropriate after an incubation period, analyte constituents bound
to the immobilized PARP homolog; or a2) immobilizing on a support
an analyte which comprises at least one possible binding partner
for a PARP molecule; b2) contacting the immobilized analyte with at
least one PARP homolog as claimed in claim 1 for which a binding
partner is sought; and c2) examining the immobilized analyte, where
appropriate after an incubation period, for binding of the PARP
homolog.
24. A method for the qualitative or quantitative determination of
nucleic acids encoding a PARP homolog as claimed in claim 1, which
comprises a) incubating a biological sample with a defined amount
of an exogenous nucleic acid as claimed in claim 6, hybridizing
under stringent conditions, determining the hybridizing nucleic
acids and, where appropriate, comparing with a standard; or b)
incubating a biological sample with a pair of oligonucleotide
primers with specificity for a PARP homolog-encoding nucleic acid,
amplifying the nucleic acid, determining the amplification product
and, where appropriate, comparing with a standard.
25. A method for the qualitative or quantitative determination of a
PARP homolog as claimed in claim 1, which comprises a) incubating a
biological sample with a binding partner specific for a PARP
homolog, b) detecting the binding partner/PARP complex and, where
appropriate, c) comparing the result with a standard.
26. The method as claimed in claim 25, wherein the binding partner
is an antibody or a binding fragment thereof, which carries a
detectable label where appropriate.
27. The method as claimed in claim 24 for diagnosing energy
deficit-mediated illnesses.
28. A method for determining the efficacy of PARP effectors, which
comprises a) incubating a PARP homolog as claimed in claim 1 with
an analyte which comprises an effector of a physiological or
pathological PARP activity; removing the effector again where
appropriate; and b) determining the activity of the PARP homolog,
where appropriate after adding substrates or cosubstrates.
29. A gene therapy composition, which comprises in a vehicle
acceptable for gene therapy a nucleic acid construct which a)
comprises an antisense nucleic acid against a coding nucleic acid
as claimed in claim 6; or b) a ribozyme against a nucleic acid as
claimed in claim 6; or c) codes for a specific PARP inhibitor.
30. A pharmaceutical composition comprising, in a pharmaceutically
acceptable vehicle, at least one PARP protein as claimed in claim
1, at least one PARP binding partner as claimed in claim 5 or at
least one coding nucleotide sequence as claimed in claim 6.
31. The use of low molecular weight PARP binding partners as
claimed in claim 5 for the manufacture of a pharmaceutical agent
for the diagnosis or therapy of pathological states in the
development and/or progress of which at least one PARP protein, or
a polypeptide derived therefrom, is involved.
32. The use of low molecular weight PARP binding partners as
claimed in claim 5 for the manufacture of a pharmaceutical agent
for the diagnosis or therapy of pathological states mediated by an
energy deficit.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This is a continuation of U.S. patent application Ser. No.
12/778,692, filed on May 12, 2010, which is a continuation of U.S.
patent application Ser. No. 09/701,586, filed on Nov. 30, 2000, now
U.S. Pat. No. 7,754,459, which is a U.S. national stage entry of
International Patent Application No. PCT/EP1999/003889, filed on
Jun. 4, 1999, which claims priority to German Patent Application
No. 19908837.3, filed on Mar. 1, 1999, and German Patent
Application No. 19825213.7, filed on Jun. 5, 1998, the entire
contents of all of which are fully incorporated herein by
reference.
[0002] The present invention relates to novel poly (ADP-ribose)
polymerase (PARP) genes and to the proteins derived therefrom;
antibodies with specificity for the novel proteins; pharmaceutical
and gene therapy compositions which comprise products according to
the invention; methods for the analytical determination of the
proteins and nucleic acids according to the invention; methods for
identifying effectors or binding partners of the proteins according
to the invention; methods for determining the activity of such
effectors and use thereof for the diagnosis or therapy of
pathological states.
[0003] In 1966, Chambon and co-workers discovered a 116 kD enzyme
which was characterized in detail in subsequent years and is now
called PARP (EC 2.4.2.30) (poly(adenosine-5'-diphosphoribose)
polymerase), PARS (poly(adenosine-5'-diphosphoribose) synthase) or
ADPRT (adenosine-5'-diphosphoribose transferase). In the plant
kingdom (Arabidopsis thaliana) a 72 kD (637 amino acids) PARP was
found in 1995 (Lepiniec L. et al., FEES Lett 1995; 364(2):
103-8). It was not clear whether this shorter form of PARP is a
plant-specific individuality or an artefact ("splice" variant or
the like). The 116 kD PARP enzyme has to date been unique in
animals and in man in its activity, which is described below. It is
referred to as PARP1 below to avoid ambiguity.
[0004] The primary physiological function of PARP 1 appears to be
its involvement in a complex repair mechanism which cells have
developed to repair DNA strand breaks. The primary cellular
response to a DNA strand break appears moreover to consist of
PARP1-catalyzed synthesis of poly(ADP-ribose) from NAD.sup.+ (cf.
De Murcia, G. et al. (1994) TIBS, 19, 172).
[0005] PARP 1 has a modular molecular structure. Three main
functional elements have been identified to date: an N-terminal 46
kD DNA binding domain; a central 22 kD automodification domain to
which poly(ADP-ribose) becomes attached, with the PARP 1 enzyme
activity decreasing with increasing elongation; and a C-terminal 54
kD NAD.sup.+ binding domain. A leucine zipper region has been found
within the automodification domain, indicating possible
protein-protein interactions, only in the PARP from Drosophila. All
PARPs known to date are presumably active as homodimers.
[0006] The high degree of organization of the molecule is reflected
in the strong conservation of the amino acid sequence. Thus, 62%
conservation of the amino acid sequence has been found for PARP 1
from humans, mice, cattle and chickens. There are greater
structural differences from the PARP from Drosophila. The
individual domains themselves in turn have clusters of increased
conservation. Thus, the DNA binding region contains two so-called
zinc fingers as subdomains (comprising motifs of the type
CX.sub.2CX.sub.28/30HX.sub.2C), which are involved in the
Zn.sup.2+-dependent recognition of DNA single strand breaks or
single-stranded DNA overhangs (e.g. at the chromosome ends, the
telomeres). The C-terminal catalytic domain comprises a block of
about 50 amino acids (residues 859-908), which is about 100%
conserved among vertebrates (PARP "signature"). This block binds
the natural substrate NAD.sup.+ and thus governs the synthesis of
poly(ADP-ribose) (cf. de Murcia, loc.cit.). The GX.sub.3GKG motif
in particular is characteristic of PARPs in this block.
[0007] The beneficial function described above contrasts with a
pathological one in numerous diseases (stroke, myocardial infarct,
sepsis etc.). PARP is involved in cell death resulting from
ischemia of the brain (Choi, D. W., (1997) Nature Medicine, 3, 10,
1073), of the myocardium (Zingarelli, B., et al (1997),
Cardiovascular Research, 36, 205) and of the eye (Lam, T. T.
(1997), Res. Comm. in Molecular Pathology and Pharmacology, 95, 3,
241). PARP activation induced by inflammatory mediators has also
been observed in septic shock (Szabo, C., et al. (1997), Journal of
Clinical Investigation, 100, 3, 723). In these cases, activation of
PARP is accompanied by extensive consumption of NAD.sup.+. Since
four moles of ATP are consumed for the biosynthesis of one mole of
NAD.sup.+, the cellular energy supply decreases drastically. The
consequence is cell death.
[0008] PARP1 inhibitors described in the abovementioned specialist
literature are nicotinamide and 3-aminobenzamide.
3,4-Di-hydro-5-[4-(1-piperidinyl)butoxy]-1 (2H)-isoquinolone is
disclosed by Takahashi, K., et al (1997), Journal of Cerebral Blood
Flow and Metabolism 17, 1137. Further inhibitors are described, for
example, in Banasik, M., et al. (1992) J. Biol. Chem., 267, 3, 1569
and Griffin, R. J., et al. (1995), Anti-Cancer Drug Design, 10,
507.
[0009] High molecular weight binding partners described for human
PARP1 include the base excision repair (BER) protein XRCC1 (X-ray
repair cross-complementing 1) which binds via a zinc finger motif
and a BRCT (BRCA1 C-terminus) module (amino acids 372-524) (Masson,
M., et al., (1998) Molecular and Cellular Biology, 18, 6,
3563).
[0010] It is an object of the present invention, because of the
diverse physiological and pathological functions of PARP, to
provide novel PARP homologs. The reason for this is that the
provision of homologous PARPs would be particularly important for
developing novel targets for drugs, and novel drugs, in order to
improve diagnosis and/or therapy of pathological states in which
PARP, PARP homologs or substances derived therefrom are
involved.
[0011] We have found that this object is achieved by providing PARP
homologs, preferably derived from human and non-human mammals,
having an amino acid sequence which has
a) a functional NAD.sup.+ binding domain, i.e. a PARP "signature"
sequence having the characteristic GX.sub.3GKG motif; and b)
especially in the N-terminal sequence region, i.e. in the region of
the first 200, such as, for example, in the region of the first
100, N-terminal amino acids, no PARP zinc finger sequence motifs of
the general formula
TABLE-US-00002 CX.sub.2CX.sub.mHX.sub.2C
in which m is an integral value from 28 or 30, and the X radicals
are, independently of one another, any amino acid; and the
functional equivalents thereof
[0012] Since the PARP molecules according to the invention
represent in particular functional homologs, they naturally also
have a poly(ADP-ribose)-synthesizing activity. The NAD binding
domain essentially corresponds to this activity and is localized to
the C terminus.
[0013] Thus an essential characteristic of the PARPs according to
the invention is the presence of a functional NAD.sup.+ binding
domain (PARP signature) which is located in the C-terminal region
of the amino acid sequence (i.e. approximately in the region of the
last 400, such as, for example, the last 350 or 300, C-terminal
amino acids), in combination with an N-terminal sequence having no
zinc finger motifs. Since the zinc finger motifs in known PARPs
presumably contribute to recognition of the DNA breakages, it is to
be assumed that the proteins according to the invention do not
interact with DNA or do so in another way. It has been demonstrated
by appropriate biochemical tests that the PARP2 according to the
invention can be activated by `activated DNA` (i.e. DNA after
limited DNaseI digestion). It can be concluded from this further
that the PARP2 according to the invention has DNA binding
properties. However, the mechanism of the DNA binding and enzyme
activation differs between the PARPs according to the invention and
PARP1. Its DNA binding and enzyme activation is, as mentioned,
mediated by a characteristic zinc finger motif. No such motifs are
present in the PARPs according to the invention. Presumably these
properties are mediated by positively charged amino acids in the
N-terminal region of the PARPs according to the invention. Since
the `activated DNA` (i.e. for example DNA after limited treatment
with DNaseI) has a large number of defects (single strand breaks,
single strand gaps, single-stranded overhangs, double strand breaks
etc.), it is possible that although PARP1 and the PARPs according
to the invention are activated by the same `activated DNA`, it is
by a different subpopulation of defects (e.g. single strand gaps
instead of single strand breaks).
[0014] The functional NAD.sup.+ binding domain (i.e. catalytic
domain) binds the substrate for poly-(ADP-ribose) synthesis.
Consistent with known PARPs, the sequence motif
GX.sup.1X.sup.2X.sup.3GKG, in which G is glycine, K is lysine, and
X.sup.1, X.sup.2 and X.sup.3 are, independently of one another, any
amino acid, is present in particular. However, as shown,
surprisingly, by comparison of the amino acid sequences of the
NAD.sup.+ binding domains of PARP molecules according to the
invention with previously disclosed human PARP1, the sequences
according to the invention differ markedly from the known sequence
for the NAD.sup.+ binding domain.
[0015] A group of PARP molecules which is preferred according to
the invention preferably has the following general sequence motif
in the catalytic domain in common:
TABLE-US-00003 (SEQ ID NO: 11) PX.sub.n(S/T)GX.sub.3GKGIYFA, in
particular (SEQ ID NO: 12)
(S/T)XGLR(I/V)XPX.sub.n(S/T)GX.sub.3GKGIYFA/ preferably (SEQ ID NO:
13)
LLWHG(S/T)X.sub.7IL(S/T)XGLR(I/V)XPX.sub.n(S/T)GX.sub.3GKGIYFAX.sub.3SKS
AXY
in which (S/T) describes the alternative occupation of this
sequence position by S or T, (I/V) describes the alternative
occupation of this sequence position by I or V, and n is an
integral value from 1 to 5, and the X radicals are, independently
of one another, any amino acid. The last motif is also referred to
as the "PARP signature" motif.
[0016] The automodification domain is preferably likewise present
in the PARPs according to the invention. It can be located, for
example, in the region from about 100 to 200 amino acids in front
of the N-terminal end of the NAD.sup.+ binding domain.
[0017] PARP homologs according to the invention may additionally
comprise, N-terminally of the NAD.sup.+ binding domain (i.e. about
30 to about 80 amino acids closer to the N terminus), a leucine
zipper-like sequence motif of the general formula
TABLE-US-00004 (SEQ ID NO: 14) (L/V)X.sub.6LX.sub.6LX.sub.6L
in which
[0018] (L/V) represents the alternative occupation of this sequence
position by L or V, and the X radicals are, independently of one
another, any amino acid. The leucine zipper motifs observed
according to the invention differ distinctly in position from those
described for PARP from Drosophila. Leucine zippers may lead to
homodimers (two PARP molecules) or heterodimers (one PARP molecule
with a binding partner differing therefrom).
[0019] The PARP homologs according to the invention preferably
additionally comprise, N-terminally of the abovementioned leucine
zipper-like sequence motifs, i.e. about 10 to 250 amino acid
residues closer to the N terminus, at least another one of the
following part-sequence motifs:
TABLE-US-00005 (motif 1; SEQ ID NO: 15)
LX.sub.9NX.sub.2YX.sub.2QLLX(D/E)X.sub.bWGRVG, (motif 2; SEQ ID NO:
16) AX.sub.3FXKX.sub.4KTXNXWX.sub.5FX.sub.3PXK, (motif 3; SEQ ID
NO: 17) QXL(I/L)X.sub.2IX.sub.9MX.sub.10PLGKLX.sub.3QIX.sub.6L,
(motif 4; SEQ ID NO: 18) FYTXIPHXFGX.sub.3 PP, and (motif 5; SEQ ID
NO: 19) KX.sub.3LX.sub.2LXDIEXAX.sub.2L,
in which (D/E) describes the alternative occupation of this
sequence position by D or E, (I/L) describes the alternative
occupation of this sequence position by I or L, b is the integral
value 10 or 11, and the X radicals are, independently of one
another, any amino acid. It is most preferred for these motifs 1 to
5 all to be present in the stated sequence, with motif 1 being
closest to the N terminus.
[0020] The abovementioned PARP signature motif is followed in the
proteins according to the invention by at least another one of the
following motifs:
TABLE-US-00006 (motif 6; SEQ ID NO: 20) GX.sub.3LXEVALG (motif 7;
SEQ ID NO: 21) GX.sub.2SX.sub.4GX.sub.3PX.sub.aLXGX.sub.2V and
(motif 8; SEQ ID NO: 22) E(Y/F)X.sub.2YX.sub.3QX.sub.4YLL
in which (Y/F) describes the alternative occupation of this
sequence position by Y or F, a is equal to 7 to 9 and X is in each
case any amino acid. It is most preferred for the three C-terminal
motifs all to be present and in the stated sequence, with motif 8
being closest to the C terminus.
[0021] A preferred PARP structure according to the invention may be
described schematically as follows:
Motifs 1 to 5/PARP signature/motifs 6 to 8 or motifs 1 to 5/leucine
zipper/PARP signature/motifs 6 to 8
[0022] it being possible for further amino acid residues, such as,
for example, up to 40, to be arranged between the individual motifs
and for further amino acid residues, such as, for example, up to
80, to be arranged at the N terminus and/or at the C terminus.
[0023] PARP homologs which are particularly preferred according to
the invention are the proteins human PARP2, human PARP3, mouse
PARP3 and the functional equivalents thereof. The protein referred
to as human PARP2 comprises 570 amino acids (cf. SEQ ID NO:2). The
protein referred to as human PARP3 possibly exists in two forms.
Type 1 comprises 533 amino acids (SEQ ID NO:4) and type 2 comprises
540 amino acids (SEQ ID NO:6). The forms may arise through
different initiation of translation. The protein referred to as
mouse PARP3 exists in two forms which differ from one another by a
deletion of 5 amino acids (15 bp). Type 1 comprises 533 amino acids
(SEQ ID NO: 8) and type 2 comprises 528 amino acids (SEQ ID NO:10).
The PARP-homologs of the present invention differ in their
sequences significantly over said PARP protein of Arabidopsis
thaliana (see above). For example, PARP2 and PARP3 do not comprise
the plant PARP specific peptide sequence AAVLDQWIPD, corresponding
to amino acid residues 143 to 152 of the Arabidopsis protein.
[0024] The invention further relates to the binding partners for
the PARP homologs according to the invention. These binding
partners are preferably selected from
a) antibodies and fragments such as, for example, Fv, Fab,
Flab').sub.2, thereof b) protein-like compounds which interact, for
example via the above leucine zipper region or another sequence
section, with PARP, and c) low molecular weight effectors which
modulate a biological PARP function such as, for example, the
catalytic PARP activity, i.e. NAD+-consuming ADP ribosylation, or
the binding to an activator protein or to DNA.
[0025] The invention further relates to nucleic acids
comprising
a) a nucleotide sequence coding for at least one PARP homolog
according to the invention, or the complementary nucleotide
sequence thereof; b) a nucleotide sequence which hybridizes with a
sequence as specified in a), preferably under stringent conditions;
or c) nucleotide sequences which are derived from the nucleotide
sequences defined in a) and b) through the degeneracy of the
genetic code.
[0026] Nucleic acids which are suitable according to the invention
comprise in particular at least one of the partial sequences which
code for the abovementioned amino acid sequence motifs.
[0027] Nucleic acids which are preferred according to the invention
comprise nucleotide sequences as shown in SEQ ID NO: 1 and 3, and,
in particular, partial sequences thereof which are characteristic
of PARP homologs according to the invention, such as, for example,
nucleotide sequences comprising
a) nucleotides +3 to +1715 shown in SEQ ID NO:1; b) nucleotides
+242 to +1843 shown in SEQ ID NO:3; c) nucleotides +221 to +1843
shown in SEQ ID NO:5; d) nucleotides +112 to +1710 shown in SEQ ID
NO:7; or e) nucleotides +1 to +1584 shown in SEQ ID NO:9 or partial
sequences of a), b), c), d) and e) which code for the
abovementioned characteristic amino acid sequence motifs of the
PARP homologs according to the invention.
[0028] The invention further relates to expression cassettes which
comprise at least one of the above-described nucleotide sequences
according to the invention under the genetic control of regulatory
nucleotide sequences. These can be used to prepare recombinant
vectors according to the invention, such as, for example, viral
vectors or plasmids, which comprise at least one expression
cassette according to the invention.
[0029] Recombinant microorganisms according to the invention are
transformed with at least one of the abovementioned vectors.
[0030] The invention also relates to transgenic mammals transfected
with a vector according to the invention.
[0031] The invention further relates to an in vitro detection
method, which can be carried out homogeneously or heterogeneously,
for PARP inhibitors, which comprises
a) incubating an unsupported or supported poly-ADP-ribosylatable
target with a reaction mixture comprising [0032] a1) a PARP homolog
according to the invention; [0033] a2) a PARP activator; and [0034]
a3) a PARP inhibitor or an analyte in which at least one PARP
inhibitor is suspected; b) carrying out the polyADP ribosylation
reaction; and c) determining the polyADP ribosylation of the target
qualitatively or quantitatively.
[0035] The detection method is preferably carried out by
preincubating the PARP homolog with the PARP activator and the PARP
inhibitor or an analyte in which at least one PARP inhibitor is
suspected, for example for about 1-30 minutes, before carrying out
the poly-ADP ribosylation reaction.
[0036] After activation by DNA with single strand breaks (referred
to as "activated DNA" according to the invention), PARP poly-ADP
ribosylates a large number of nuclear proteins in the presence of
NAD. These proteins include, on the one hand, PARP itself, but also
histones etc.
[0037] The poly-ADP-ribosylatable target preferably used in the
detection method is a histone protein in its native form or a
poly-ADP-ribosylatable equivalent derived therefrom. A histone
preparation supplied by Sigma (SIGMA, catalogue No. H-7755; histone
type II-AS from calf thymus, Luck, J. M., et al., J. Biol. Chem.,
233, 1407 (1958), Satake K., et al., J. Biol. Chem, 235, 2801
(1960)) was used by way of example. It is possible in principle to
use all types of proteins or parts thereof amenable to
poly-ADP-ribosylation by PARP. These are preferably nuclear
proteins, e.g. histones, DNA polymerase, telomerase or PARP itself.
Synthetic peptides derived from the corresponding proteins can also
act as target.
[0038] In the ELISA according to the invention it is possible to
use amounts of histones in the range from about 0.1 .mu.g/well to
about 100 .mu.g/well, preferably about 1 .mu.g/well to about 10
.mu.g/well. The amounts of the PARP enzyme are in a range from
about 0.2 pmol/-well to about 2 nmol/well, preferably from about 2
pmol/well to about 200 pmol/well, the reaction mixture comprising
in each case 100 .mu.g/well. Reductions to smaller wells and
correspondingly smaller reaction volumes are possible.
[0039] In the HTRF assay according to the invention, identical
amounts of PARP are employed, and the amount of histone or modified
hi-stones is in the range from about 2 ng/well to about 25
.mu.g/-well, preferably about 25 ng/well to about 2.5 .mu.g/well,
the reaction mixture comprising in each case 50 .mu.l/well.
Reductions to smaller wells and correspondingly smaller reaction
volumes are possible.
[0040] The PARP activator used according to the invention is
preferably activated DNA.
[0041] Various types of damaged DNA can function as activator. DNA
damage can be produced by digestion with DNases or other
DNA-modifying enzymes (e.g. restriction endonucleases), by
irradiation or other physical methods or chemical treatment of the
DNA. It is further possible to simulate the DNA damage situation in
a targeted manner using synthetic oligonucleotides. In the assays
indicated by way of example, activated DNA from calf thymus was
employed (Sigma, product No. D4522; CAS: 91080-16-9, prepared by
the method of Aposhian and Kornberg using calf thymus DNA (SIGMA
D-1501) and deoxyribonuclease type I (D-4263). Aposhian H. V. and
Kornberg A., J. Biol. Chem., 237, 519 (1962)). The activated DNA
was used in a concentration range from 0.1 to 1000 .mu.g/ml,
preferably from 1 to 100 .mu.g/ml, in the reaction step.
[0042] The polyADP ribosylation reaction is started in the method
according to the invention by adding NAD.sup.+. The NAD
concentrations were in a range from about 0.1 .mu.M to about 10 mM,
preferably in a range from about 10 .mu.M to about 1 mM.
[0043] In the variant of the above method which can be carried out
heterogeneously, the polyADP ribosylation of the supported target
is determined using anti-poly(ADP-ribose) antibodies. To do this,
the reaction mixture is separated from the supported target, washed
and incubated with the antibody. This antibody can itself be
labeled. However, as an alternative for detecting bound
anti-poly(ADP-ribose) antibody a labeled secondary antibody or a
corresponding labeled antibody fragment may be applied. Suitable
labels are, for example, radiolabeling, chromophore- or
fluoro-phore-labeling, biotinylation, chemiluminescence labeling,
labeling with paramagnetic material or, in particular, enzyme
labels, e.g. with horseradish peroxidase. Appropriate detection
techniques are generally known to the skilled worker.
[0044] In the variant of the above process which can be carried out
homogeneously, the unsupported target is labeled with an acceptor
fluorophore. The target preferably used in this case is
biotinylated histone, the acceptor fluorophore being coupled via
avidin or streptavidin to the biotin groups of the histone.
Particularly suitable as acceptor fluorophore are phycobiliproteins
(e.g. phycocyanins, phycoerythrins), e.g. R-phycocyanin (R-PC),
allophycocyanin (APC), R-phycoerythrin (R-PE), C-phycocyanin
(C-PC), B-phycoerythrin (B-PE) or their combinations with one
another or with fluorescent dyes such as Cy5, Cy7 or Texas Red
(Tandem system) (Thammapalerd, N. et al., Southeast Asian Journal
of Tropical Medicine & Public Health, 27(2): 297-303 (1996);
Kronick, M. N. et al., Clinical Chemistry, 29(9), 1582-1586 (1986);
Hicks, J. M., Human Pathology, 15(2), 112-116 (1984)). The dye
XL665 used in the examples is a crosslinked allophycocyanin
(Glazer, A. N., Rev. Microbiol., 36, 173-198 (1982); Kronick, M.
N., J. Imm. Meth., 92, 1-13 (1986); Maccoll, R. et al.,
Phycobiliproteins, CRC Press, Inc., Boca Raton, Fla. (1987);
Maccoll, R. et al., Arch. Biochem. Biophys., 208(1), 42-48
(1981)).
[0045] It is additionally preferred in the homogeneous method to
determine the polyADP ribosylation of the unsupported target using
anti-poly(ADP-ribose) antibody which is labeled with a donor
fluorophore which is able to transfer energy to the acceptor
fluorophore when donor and acceptor are close in space owing to
binding of the labeled antibody to the polyADP-ribosylated
hi-stone. A europium cryptate is preferably used as donor
fluoro-phore for the anti-poly(ADP-ribose) antibody.
[0046] Besides the europium cryptate used, other compounds are also
possible as potential donor molecules. This may entail, on the one
hand, modification of the cryptate cage. Replacement of the
europium by other rare earth metals such as terbium is also
conceivable. It is crucial that the fluorescence has a long
duration to guarantee the time delay (Lopez, E. et al., Clin. Chem.
39/2, 196-201 (1993); U.S. Pat. No. 5,534,622).
[0047] The detection methods described above are based on the
principle that there is a correlation between the PARP activity and
the amount of ADP-ribose polymers formed on the histones. The assay
described herein makes it possible to quantify the ADP-ribose
polymers using specific antibodies in the form of an ELISA and an
HTRF (homogenous time-resolved fluorescence) assay. Specific
embodiments of these two assays are described in detail in the
following examples.
[0048] The developed HTRF (homogeneous time-resolved fluorescence)
assay system measures the formation of poly(ADP-ribose) on histones
using specific antibodies. In contrast to the ELISA, this assay is
carried out in homogeneous phase without separation and washing
steps. This makes a higher sample throughput and a smaller
susceptibility to errors possible. HTRF is based on the
fluorescence resonance energy transfer (FRET) between two
fluorophores. In a FRET assay, an excited donor fluorophore can
transfer its energy to an acceptor fluorophore when the two are
close to one another in space. In HTRF technology, the donor
fluorophore is a europium cryptate [(Eu)K] and the acceptor is
XL665, a stabilized allophycocyanin. The europium cryptate is based
on studies by Jean Marie Lehn (Strasbourg) (Lopez, E. et al., Clin.
Chem. 39/2, 196-201 (1993); U.S. Pat. No. 5,534,622).
[0049] In a homogeneous assay, all the components are also present
during the measurement. Whereas this has advantages for carrying
out the assay (rapidity, complexity), it is necessary to preclude
interference by assay components (inherent fluorescence, quenching
by dyes etc.). HTRF precludes such interference by time-delayed
measurement at two wavelengths (665 nm, 620 nm). The HTRF has a
very long decay time and time-delayed measurement is therefore
possible. There is no longer any interference from short-lived
background fluorescence (e.g. from assay components or inhibitors
of the substance library). In addition, measurement is always
carried out at two wavelengths in order to compensate for quench
effects of colored substances. HTRF assays can be carried out, for
example, in 96- or 384-well microtiter plate format and are
evaluated using a discovery HTRF microplate analyzer (Canberra
Packard).
[0050] Also provided according to the invention are the following
in vitro screening methods for binding partners for PARP, in
particular for a PARP homolog according to the invention.
[0051] A first variant is carried out by
a1) immobilizing at least one PARP homolog on a support; b1)
contacting the immobilized PARP homolog with an analyte in which at
least one binding partner is suspected; and c1) determining, where
appropriate after an incubation period, analyte constituents bound
to the immobilized PARP homolog.
[0052] A second variant entails
a2) immobilizing on a support an analyte which comprises at least
one possible binding partner for the PARP homolog; b2) contacting
the immobilized analyte with at least one PARP homolog for which a
binding partner is sought; and c3) examining the immobilized
analyte, where appropriate after an incubation period, for binding
of the PARP homolog.
[0053] The invention also relates to a method for the qualitative
or quantitative determination of a nucleic acid encoding a PARP
homolog, which comprises
a) incubating a biological sample with a defined amount of an
exogenous nucleic acid according to the invention (e.g. with a
length of about 20 to 500 bases or longer), hybridizing, preferably
under stringent conditions, determining the hybridizing nucleic
acids and, where appropriate, comparing with a standard; or b)
incubating a biological sample with a defined amount of
oligonucleotide primer pairs with specificity for a PARP
homolog-encoding nucleic acid, amplifying the nucleic acid,
determining the amplification product and, where appropriate,
comparing with a standard.
[0054] The invention further relates to a method for the
qualitative or quantitative determination of a PARP homolog
according to the invention, which comprises
a) incubating a biological sample with at least one binding partner
specific for a PARP homolog, b) detecting the binding partner/PARP
complex and, where appropriate, c) comparing the result with a
standard.
[0055] The binding partner in this case is preferably an anti-PARP
antibody or a binding fragment thereof, which carries a detectable
label where appropriate.
[0056] The determination methods according to the invention for
PARP, in particular for PARP homologs and for the coding nucleic
acid sequences thereof, are suitable and advantageous for
diagnosing sepsis- or ischemia-related tissue damage, in particular
strokes, myocardial infarcts, diabetes or septic shock.
[0057] The invention further comprises a method for determining the
efficacy of PARP effectors, which comprises
a) incubating a PARP homolog according to the invention with an
analyte which comprises an effector of a physiological or
pathological PARP activity; removing the effector again where
appropriate; and b) determining the activity of the PARP homolog,
where appropriate after adding substrates or cosubstrates.
[0058] The invention further relates to gene therapy compositions
which comprise in a vehicle acceptable for gene therapy a nucleic
acid construct which
a) comprises an antisense nucleic acid against a coding nucleic
acid according to the invention; or b) a ribozyme against a
noncoding nucleic acid according to the invention; or c) codes for
a specific PARP inhibitor.
[0059] The invention further relates to pharmaceutical compositions
comprising, in a pharmaceutically acceptable vehicle, at least one
PARP protein according to the invention, at least one PARP binding
partner according to the invention or at least one coding
nucleotide sequence according to the invention.
[0060] Finally, the invention relates to the use of binding
partners of a PARP homolog for the diagnosis or therapy of
pathological states in the development and/or progress of which at
least one PARP protein, in particular a PARP homolog according to
the invention, or a polypeptide derived therefrom, is involved. The
binding partner used can be, for example, a low molecular weight
binding partner whose molecular weight can be, for example, less
than about 2000 dalton or less than about 1000 dalton.
[0061] The invention additionally relates to the use of PARP
binding partners for the diagnosis or therapy of pathological
states mediated by an energy deficit. An energy deficit for the
purpose of the present invention is, in particular, a cellular
energy deficit which is to be observed in the unwell patient
systemically or in individual body regions, organs or organ
regions, or tissues or tissue regions. This is characterized by an
NAD and/or ATP depletion going beyond (above or below) the
physiological range of variation of the NAD and/or ATP level and
mediated preferably by a protein with PARP activity, in particular
a PARP homolog according to the invention, or a polypeptide derived
therefrom.
[0062] "Energy deficit-mediated disorders" for the purpose of the
invention additionally comprise those in which tissue damage is
attributable to cell death resulting from necrosis or apoptosis.
The methods according to the invention are suitable for treating
and preventing tissue damage resulting from cell damage due to
apoptosis or necrosis; damage to nerve tissue due to ischemias
and/- or reperfusion; neurological disorders; neurodegenerative
disorders; vascular stroke; for treating and preventing
cardiovascular disorders; for treating other disorders or
conditions such as, for example, age-related macular degeneration,
AIDS or other immunodeficiency disorders; arthritis;
atherosclerosis; cachexia; cancer; degenerative disorders of the
skeletal muscles; diabetes; cranial trauma; inflammatory disorders
of the gastrointestinal tract such as, for example, Crohn's
disease; muscular dystrophy; osteoarthritis; osteoporosis; chronic
and/or acute pain; kidney failure; retinal ischemia; septic shock
(such as, for example, endotoxin shock); aging of the skin or aging
in general; general manifestations of aging. The methods according
to the invention can additionally be employed for extending the
life and the pro-liferative capacity of body cells and for
sensitizing tumor cells in connection with irradiation therapy.
[0063] The invention particularly relates to the use of a PARP
binding partner as defined above for the diagnosis or therapy
(acute or prophylactic) of pathological states mediated by energy
deficits and selected from neurodegenerative disorders, or tissue
damage caused by sepsis or ischemia, in particular of neurotoxic
disturbances, strokes, myocardial infarcts, damage during or after
infarct lysis (e.g. with TPA, Reteplase or mechanically with laser
or Rotablator) and of microinfarcts during and after heart valve
replacement, aneurysm resections and heart transplants, trauma to
the head and spinal cord, infarcts of the kidney (acute kidney
failure, acute renal insufficiency or damage during and after
kidney transplant), damages of skeletal muscle, infarcts of the
liver (liver failure, damage during or after a liver transplant),
peripheral neuropathies, AIDS dementia, septic shock, diabetes,
neurodegenerative disorders occuring after ischemia, trauma
(craniocerebral trauma), massive bleeding, subarachnoid hemorrhages
and stroke, as well as neurodegenerative disorders like Alzheimer's
disease, multi-infarct dementia, Huntington's disease, Parkinson's
disease, amyotrophic lateral sclerosis, epilepsy, especially of
generalized epileptic seizures such as petit mal and tonoclonic
seizures and partial epileptic seizures, such as temporal lobe, and
complex partial seizures, kidney failure, also in the chemotherapy
of tumors and prevention of metastasis and for the treatment of
inflammations and rheumatic disorders, e.g. of rheumatoid
arthritis; further for the treatment of revascularization of
critically narrowed coronary arteries and critically narrowed
peripheral arteries, e.g. leg arteries.
[0064] "Ischemia" comprises for the purposes of the invention a
localized undersupply of oxygen to a tissue, caused by blockage of
arterial blood flow. Global ischemia occurs when the blood flow to
the entire brain is interrupted for a limited period. This may be
caused, for example, by cardiac arrest. Focal ischemia occurs when
part of the brain is cut off from its normal blood supply. Focal
ischemia may be caused by thromboembolic closure of a blood vessel,
by cerebral trauma, edemas or brain tumor. Even transient ischemias
can lead to wideranging neuronal damage. Although damage to "nerve
tissue" may occur days or weeks after the start of the ischemia,
some permanent damage (e.g. necrotic cell death) occurs in the
first few minutes after interruption of the blood supply. This
damage is caused, for example, by the neurotoxicity of glutamate
and follows secondary reperfusion, such as, for example, release of
free radicals (e.g. oxygen free radicals, NO free radicals).
Ischemias may likewise occur in other organs and tissues such as,
for example, in the heart (myocardial infarct and other
cardiovascular disorders caused by occlusion of the coronary
arteries) or in the eye (ischemia of the retina).
[0065] The invention additionally relates to the use of an
effective therapeutic amount of a PARP binding partner for
influencing neuronal activity. "Neuronal activity" for the purposes
of the invention may consist of stimulation of damaged neurons,
promotion of neuronal regeneration or treatment of neuronal
disorders.
[0066] "Neuronal damage" for the purposes of the invention
comprises every type of damage to "nerve tissue" and every physical
or mental impairment or death resulting from this damage. The cause
of the damage may be, for example, metabolic, toxic, chemical or
thermal in nature and includes by way of example ischemias,
hypoxias, trauma, cerebrovascular damage, operations, pressure,
hemorrhages, irradiation, vasospasms, neurodegenerative disorders,
infections, epilepsy, perception disorders, disturbances of
glutamate metabolism and the secondary effects caused thereby.
[0067] "Nerve tissue" for the purposes of the invention comprises
the various components forming the nervous system, consisting of,
inter alia, neurons, glia cells, astrocytes, Schwann cells, the
vascular system inside and for supplying, the CNS, brain, brain
stem, spinal cord, peripheral nervous system etc.
[0068] "Neuroprotective" for the purposes of the invention
comprises the reduction, the cessation, the slowing down or the
improvement of neuronal damage and the protection, the restoration
and the regeneration of nerve tissue which was exposed to neuronal
damage.
[0069] "Prevention of neurodegenerative disorders" includes the
possibility of preventing, slowing down and improving
neurodegenerative disorders in people for whom such a disorder has
been diagnosed or who are included in appropriate risk groups for
these neurodegenerative disorders. Treatments for people already
suffering from symptoms of these disorders are likewise meant.
[0070] "Treatment" for the purposes of the invention comprises
(i) preventing a disorder, a disturbance or a condition in people
with a predisposition thereto; (ii) preventing a disorder, a
disturbance or a condition by slowing down its advance; and (iii)
improving a disorder, a disturbance or a condition.
[0071] Examples of "neurological disorders" which can be treated by
the methods according to the invention are neuralgias (trigeminal,
glossopharyngeal), myasthenia gravis, muscular dystrophies,
amyorophic lateral sclerosis (ALS), progressive muscular atrophy,
peripheral neuropathies caused by poisoning (e.g. lead poisoning),
Guillain-Barre syndrome, Huntington's disease, Alzheimer's disease,
Parkinson's disease, or plexus disorders. The methods according to
the invention are preferably suitable for treating neurological
disorders selected from peripheral neuropathies caused by physical
injury or illness; cranial trauma such as, for example, traumatic
brain injury; physical damage to the spinal cord; stroke associated
with brain damage, such as vascular stroke in conjunction with
hypoxia and brain damage, and cerebral reperfusion damage;
demyelinating disorders (myelopathies, Alzheimer's disease,
Parkinson's disease, Huntington's disease, amyotrophic lateral
sclerosis).
[0072] The methods according to the invention can additionally be
used for treating cardiovascular disorders. "Cardiovascular
disorders" for the purposes of the invention comprise those which
cause ischemias or are caused by ischemias or ischemia/reperfusion
of the heart. Examples are coronary vessel disorders (for example
atherosclerosis), angina pectoris, myocardial infarct,
cardiovascular damage due to cardiac arrest or bypass
operation.
[0073] The methods according to the invention can be used for
treating cancer or for sensitizing cancer cells for irradiation
therapy. The term "cancer" is to be understood in the widest sense.
Modulators of the proteins according to the invention can be used
as "anti-cancer therapy agents". For example, the methods can be
used for treating types of cancer or tumor cells, such as
ACTH-producing tumors, acute lymphatic or lymphoblastic leukemia;
acute or chronic lymphocytic leukemia; acute nonlymphocytic
leukemia; bladder cancer; brain tumors; breast cancer; cervical
carcinoma; chronic myelocytic leukemia; bowel cancer; T-zone
lymphoma; endometriosis; esophageal cancer; gall bladder cancer;
Ewing's sarcoma; head and neck cancer; cancer of the tongue;
Hodgkin's lymphoma; Kaposi's sarcoma; renal cancer; liver cancer;
lung cancer; mesothelioma; multiple myeloma; neuroblastoma;
non-Hodgkin lymphoma; osteosarcoma; ovarian carcinoma;
glioblastoma; mammary carcinoma; cervical carcinoma; prostate
cancer; pancreatic cancer; penis cancer; retinoblastoma; skin
cancer; stomach cancer; thyroid cancer; uterine carcinoma; vaginal
carcinoma; Wilm's tumor; or trophoblastoma.
[0074] "Radiosensitizer" or "irradiation sensitizer" for the
purposes of the invention relates to molecules which increase the
sensitivity of the cells in the body to irradiation with
electromagnetic radiation (for example X-rays) or speed up this
irradiation treatment. Irradiation sensitizers increase the
sensitivity of cancer cells to the toxic effects of the
electromagnetic radiation. Those disclosed in the literature
include mitomycin C, 5-bromo-deoxyuridine and metronidazole. It is
possible to use radiation with wavelengths in the range from
10.sup.-20 to 10 meters, preferably gamma rays (10.sup.-20 to
10.sup.-13 m), X-rays (10.sup.-11 to 10.sup.-9 m), ultraviolet
radiation (10 nm to 400 nm), visible light (400 nm to 700 nm),
infrared radiation (700 nm to 1 mm) and microwave radiation (1 mm
to 30 cm).
[0075] Disorders which can be treated by such a therapy are, in
particular, neoplastic disorders, benign or malignant tumors and
cancer. The treatment of other disorders using electromagnetic
radiation is likewise possible.
[0076] The present invention will now be described in more detail
with reference to the appended figures. These show:
[0077] FIG. 1 a sequence alignment of human PARP (human PARP1) and
two PARPs preferred according to the invention (human PARP2, human
PARP3, murine PARP3). Sequence agreements between human PARP1 and
human PARP2, human PARP3 or murine PARP3 are depicted within
frames. The majority sequence is indicated over the alignment. The
zinc finger motifs of human PARP1 are located in the sequence
sections corresponding to amino acid residues 21 to 56 and 125 to
162;
[0078] FIG. 2 Northern blots with various human tissues to
illustrate the tissue distribution of PARP2 and PARP3 molecules
according to the invention. Lane 1: brain; lane 2: heart; lane 3:
skeletal muscle; lane 4: colon; lane 5: thymus; lane 6: spleen;
lane 7: kidney; lane 8: liver; lane 9: intestine; lane 10:
placenta; lane 11: lung; lane 12: peripheral blood leukocytes; the
respective position of the size standard (kb) is indicated.
[0079] FIG. 3 a Northern blot with further various human tissues to
illustrate the tissue distribution of the PARP3 molecule according
to the invention. Lane 1: heart; lane 2: brain; lane 3: placenta;
lane 4: lung; lane 5: liver; lane 6: skeletal muscle; lane 7:
kidney; lane 8: pancreas; the respective position of the size
standard (kb) is indicated.
[0080] FIG. 4 a Western blot with various human tissues to
illustrate the tissue distribution of the PARP3 molecule according
to the invention at the protein level. Lane 1: heart; lane 2: lung;
lane 3: liver; lane 4: spleen; lane 5: kidney; lane 6: colon; lane
7: muscle; lane 8: brain; the respective position of the size
standard (kD) is indicated.
[0081] FIG. 5 a Western blot with various human tissues to
illustrate the tissue distribution of the PARP3 molecule according
to the invention. Lane 1: frontal cortex; lane 2: posterior cortex;
lane 3: cerebellum; lane 4: hippocampus; lane 5: olfactory bulb;
lane 6: striatum; lane 7: thalamus; lane 8: midbrain; lane 9:
entorhinal cortex; lane 10: pons; lane 11: medulla; lane 12: spinal
cord.
[0082] FIG. 6 a diagrammatic representation of the PARP assay
(ELISA)
[0083] FIG. 7 a diagrammatic representation of the PARP assay
(HTRF)
[0084] Further preferred embodiments of the invention are described
in the following sections.
[0085] PARP homologs and functional equivalents
[0086] Unless stated otherwise, for the purposes of the present
description amino acid sequences are indicated starting with the N
terminus. If the one-letter code is used for amino acids, then G is
glycine, A is alanine, V is valine, L is leucine, I is isoleucine,
S is serine, T is threonine, D is aspartic acid, N is asparagine, E
is glutamic acid, Q is glutamine, W is tryptophan, H is histidine,
R is arginine, P is praline, K is lysine, Y is tyrosine, F is
phenylalanine, C is cysteine and M is methionine.
[0087] The present invention is not confined to the PARP homologs
specifically described above. On the contrary, those homologs which
are functional equivalents thereof are also embraced. Functional
equivalents comprise both natural, such as, for example,
species-specific or organ-specific, and artificially produced
variants of the proteins specifically described herein. Functional
equivalents according to the invention differ by addition,
substitution, inversion, insertion and/or deletion of one or more
amino acid residues of human PARP2 (SEQ ID NO:2), human PARP3 (SEQ
ID NO: 4 and 6) and mouse PARP3 (SEQ ID:8 and 10), there being at
least retention of the NAD-binding function of the protein mediated
by a functional catalytic C-terminal domain. Likewise, the
poly(ADP-ribose)-producing catalytic activity should preferably be
retained. Functional equivalents also comprise where appropriate
those variants in which the region similar to the leucine zipper is
essentially retained.
[0088] It is moreover possible, for example, starting from the
sequence for human PARP2 or human PARP3 to replace certain amino
acids by those with similar physicochemical properties (bulk,
basicity, hydrophobicity, etc.). It is possible, for example, for
arginine residues to be replaced by lysine residues, valine
residues by isoleucine residues or aspartic acid residues by
glutamic acid residues. However, it is also possible for one or
more amino acids to be exchanged in sequence, added or deleted, or
several of these measures can be combined together. The proteins
which have been modified in this way from the human PARP2 or human
PARP3 sequence have at least 60%, preferably at least 75%, very
particularly preferably at least 85%, homology with the starting
sequence, calculated using the algorithm of Pearson and Lipman,
Proc. Natl. Acad. Sci (USA) 85(8), 1988, 2444-2448.
[0089] The following homologies have been determined at the amino
acid level and DNA level between human PARP1, 2 and 3 (FastA
program, Pearson and Lipman, loc. cit.):
Amino Acid Homologies:
TABLE-US-00007 [0090] Percent identity in PARP Percent identity
signature PARP1/PARP2 41.97% (517) 86% (50) PARP1/PARP3 33.81%
(565) 53.1% (49) PARP2/PARP3 35.20% (537) 53.1% (49)
[0091] Numbers in parentheses indicate the number of overlapping
amino acids.
DNA Homologies:
TABLE-US-00008 [0092] Percent identity in PARP Percent identity in
the ORF signature PARP1/PARP3 60.81% (467) 77.85% (149) PARP1/PARP3
58.81% (420) 59.02% (61) PARP2/PARP3 60.22% (269) 86.36% (22)
[0093] Numbers in parentheses indicate the number of overlapping
nucleotides.
[0094] The polypeptides according to the invention can be
classified as homologous poly(ADP-ribose) polymerases on the basis
of the great similarity in the region of the catalytic domain.
[0095] It is also essential to the invention that the novel PARP
homologs do not have conventional zinc finger motifs. This means
that these enzymes are not necessarily involved in DNA repair or
are so in a way which differs from PARP1, but are still able to
carry out their pathological mechanism (NAD.sup.+ consumption and
thus energy consumption due to ATP consumption). The strong protein
expression, particularly of PARP3, observable in the Western blot
suggests a significant role in the NAD consumption. This is
particularly important for drug development. Potential novel
inhibitors of the polymerases according to the invention can thus
inhibit the pathological functions without having adverse effects
on the desired physiological properties. This was impossible with
inhibitors against the PARPs known to date since there was always
also inhibition of the DNA repair function. The potentially
mutagenic effect of known PARP inhibitors is thus easy to
understand. It is also conceivable to design PARP inhibitors so
that they efficiently inhibit all PARP homologs with high affinity.
In this case, a potentiated effect is conceivable where
appropriate.
[0096] The PARP homolog which is preferred according to the
invention and is shown in SEQ ID NO:2 (human PARP2) can
advantageously be isolated from human brain, heart, skeletal
muscle, kidney and liver. The expression of human PARP2 in other
tissues or organs is distinctly weaker.
[0097] The PARP homolog which is preferred according to the
invention and is shown in SEQ ID NO: 4 and 6 (human PARP3) can
advantageously be isolated from human brain (in this case very
preferentially from the hippocampus), heart, skeletal muscle, liver
or kidney. The expression of human PARP3 in other tissues or
organs, such as muscle or liver, is distinctly weaker.
[0098] The skilled worker familiar with protein isolation will make
use of the combination of preparative methodologies which is most
suitable in each case for isolating natural PARPs according to the
invention from tissues or recombinantly prepared PARPs according to
the invention from cell cultures. Suitable standard preparative
methods are described, for example, in Cooper, T. G., Biochemische
Arbeitsmethoden, published by Walter de Gruyter, Berlin, N.Y. or in
Scopes, R. Protein Purification, Springer Verlag, New York,
Heidelberg, Berlin.
[0099] The invention additionally relates to PARP2 and PARP3
homologs which, although they can be isolated from other eukaryotic
species, i.e. invertebrates or vertebrates, especially other
mammals such as, for example, mice, rats, cats, dogs, pigs, sheep,
cattle, horses or monkeys, or from other organs such as, for
example the myocardium, have the essential structural and
functional properties predetermined by the PARPs according to the
invention.
[0100] In particular, the human PARP2 which can be isolated from
human brain, and its functional equivalents, are preferred agents
for developing inhibitors of neurodegenerative diseases as for
example stroke. This is because it can be assumed that drug
development based on PARP2 as indicator makes it possible to
develop inhibitors which are optimized for use in the human brain.
However, it cannot be ruled out that inhibitors developed on the
basis of PARP2 can also be employed for treating PARP-mediated
pathological states in other organs, too (see tissue distribution
of the proteins according to the invention).
[0101] PARP2 and presumably PARP3 are also, similar to PARP 1,
activated by damaged DNA, although by a presumably different
mechanism. Significance in DNA repair is conceivable. Blockade of
the PARPs according to the invention would also be beneficial in
indications such as cancer (e.g. in the radiosensitization of tumor
patients).
[0102] Another essential biological property of PARPs according to
the invention and their functional equivalents is to be seen in
their ability to bind an interacting partner. Human PARP2 and 3
differ from previously disclosed PARPs from higher eukaryotes such
as, in particular, mammals by having potential so-called leucine
zipper motifs. This is a typical motif for protein-protein
interactions. It is possible that these motifs permit modulation of
PARP activity by an interacting partner. This additional structural
element thus also provides a possible starting point for
development of PARP effectors such as, for example, inhibitors.
[0103] The invention thus further relates to proteins which
interact with PARP2 and/or 3, preferably those which bring about
their activation or inactivation.
[0104] The invention further relates to proteins which still have
the abovementioned ligand-binding activity and which can be
prepared starting from the specifically disclosed amino acid
sequences by targeted modifications.
[0105] It is possible, starting from the peptide sequence of the
proteins according to the invention, to generate synthetic peptides
which are employed, singly or in combination, as antigens for
producing polyclonal or monoclonal antibodies. It is also possible
to employ the PARP protein or fragments thereof for generating
antibodies. The invention thus also relates to peptide fragments of
PARP proteins according to the invention which comprise
characteristic partial sequences, in particular those oligo- or
polypeptides which comprise at least one of the abovementioned
sequence motifs. Fragments of this type can be obtained, for
example, by proteolytic digestion of PARP proteins or by chemical
synthesis of peptides.
Novel Specific PARP2 and PARP3 Binding Partners
[0106] Active and preferably selective inhibitors against the
proteins according to the invention were developed using the
specific assay systems described above for binding partners for
PARP2 and PARP3. These inhibitors optionally are also active vis a
vis PARP1.
[0107] Inhibitors provided according to the invention have a strong
inhibitory activity on PARP2. The K.sub.i values may in this case
be less than about 1000 nM, such as less than about 700 nM, less
than about 200 nM or less than about 30 nM, e.g. about 1 to 20
nM.
[0108] Inhibitors according to the invention may also have a
surprising selectivity for PARP2. This is shown by the
K.sub.i(PARP1): K.sub.i(PARP2) ratio for such inhibitors according
to the invention which is, for example, greater than 3 or greater
than 5, as for example greater than 10 or greater than 20.
[0109] An example which should be mentioned is
4-(N-(4-hydroxyphenyl)aminomethyl)-(2H)-dihydrophthalazine-1-one.
The preparation of this and other analogous compounds may be
performed according to Puodzhyunas et al., Pharm. Chem. J. 1973, 7,
566 or Mazkanowa et al., Zh. Obshch. Khim., 1958, 28, 2798, or
Mohamed et al., Ind. J. Chem. B., 1994, 33, 769 each incorporated
by reference.
[0110] The above identified compound shows a Ki value of 113 nM for
PARP2 and is eight times more selective for PARP2 than for
PARP3.
Nucleic Acids Coding for PARP Homologs:
[0111] Unless stated otherwise, nucleotide sequences are indicated
in the present description from the 5' to the 3' direction.
[0112] The invention further relates to nucleic acid sequences
which code for the abovementioned proteins, in particular for those
having the amino acid sequence depicted in SEQ ID NO: 2, 4, 6, 8
and 10, but without being restricted thereto. Nucleic acid
sequences which can be used according to the invention also
comprise allelic variants which, as described above for the amino
acid sequences, are obtainable by deletion, inversion, insertion,
addition and/or substitution of nucleotides, preferably of
nucleotides shown in SEQ ID NO: 1, 3, 7 and 9, but with essential
retention of the biological properties and the biological activity
of the corresponding gene product. Nucleotide sequences which can
be used are obtained, for example, by nucleotide substitutions
causing silent (without alteration of the amino acid sequence) or
conservative amino acid changes (exchange of amino acids of the
same size, charge, polarity or solubility).
[0113] Nucleic acid sequences according to the invention also
embrace functional equivalents of the genes, such as eukaryotic
homologs for example from invertebrates such as Caenorhabditis or
Drosophila, or vertebrates, preferably from the mammals described
above. Preferred genes are those from vertebrates which code for a
gene product which has the properties essential to the invention as
described above.
The Nucleic Acids According to the Invention can be Obtained in a
Conventional Way by Various Routes:
[0114] For example, a genomic or a cDNA library can be screened for
DNA which codes for a PARP molecule or a part thereof. For example,
a cDNA library obtained from human brain, heart or kidney can be
screened with a suitable probe such as, for example, a labeled
single-stranded DNA fragment which corresponds to a partial
sequence of suitable length selected from SEQ ID NO: 1 or 3, or
sequence complementary thereto. For this purpose, it is possible,
for example, for the DNA fragments of the library which have been
transferred into a suitable cloning vector to be, after
transformation into a bacterium, plated out on agar plates. The
clones can then be transferred to nitrocellulose filters and, after
denaturation of the DNA, hybridized with the labeled probe.
Positive clones are then isolated and characterized.
[0115] The DNA coding for PARP homologs according to the invention
or partial fragments can also be synthesized chemically starting
from the sequence information contained in the present application.
For example, it is possible for this purpose for oligonucleotides
with a length of about 100 bases to be synthesized and sequentially
ligated in a manner known per se by, for example, providing
suitable terminal restriction cleavage sites.
[0116] The nucleotide sequences according to the invention can also
be prepared with the aid of the polymerase chain reaction (PCR).
For this, a target DNA such as, for example, DNA from a suitable
full-length clone is hybridized with a pair of synthetic
oligonucleotide primers which have a length of about 15 bases and
which bind to opposite ends of the target DNA. The sequence section
lying between them is then filled in with DNA polymerase.
Repetition of this cycle many times allows the target DNA to be
amplified (cf. White et al. (1989), Trends Genet. 5, 185).
[0117] The nucleic acid sequences according to the invention are
also to be understood to include truncated sequences,
single-stranded DNA or RNA of the coding and noncoding,
complementary DNA sequence, mRNA sequences and cDNAs derived
therefrom.
[0118] The invention further embraces nucleotide sequences
hybridizing with the above sequences under stringent conditions.
Stringent hybridization conditions for the purpose of the present
invention exist when the hybridizing sequences have a homology of
about 70 to 100%, such as, for example about 80 to 100% or 90 to
100% (preferably in an amino acid section of at least about 40,
such as, for example, about 50, 100, 150, 200, 400 or 500 amino
acids).
[0119] Stringent conditions for the screening of DNA, in particular
cDNA banks, exist, for example, when the hybridization mixture is
washed with 0.1.times.SSC buffer (20.times.SSC buffer=3M NaCl, 0.3M
sodium citrate, pH 7.0) and 0.1% SDS at a temperature of about
60.degree. C.
[0120] Northern blot analyses are analyses are washed under
stringent conditions with 0.1.times.SSC, 0.1% SDS at a temperature
of about 65.degree. C., for example.
Nucleic Acid Derivatives and Expression Constructs:
[0121] The nucleic acid sequences are also to be understood to
include derivatives such as, for example, promoter variants or
alternative splicing variants. The promoters operatively linked
upstream of the nucleotide sequences according to the invention may
moreover be modified by nucleotide addition(s) or substitution(s),
inversion(s), insertion(s) and/or deletion(s), but without
impairing the functionality or activity of the promoters. The
promoters can also have their activity increased by modifying their
sequence, or be completely replaced by more effective promoters
even from heterologous organisms. The promoter variants described
above are used to prepare expression cassettes according to the
invention.
SPECIFIC EXAMPLES OF HUMAN PARP2 SPLICING VARIANTS WHICH MAY BE
MENTIONED ARE
[0122] Variant human PARP2a: Deletion of base pairs 766 to 904 (cf.
SEQ ID NO:1). This leads to a frame shift with a new stop codon
("TAA" corresponding to nucleotides 922 to 924 in SEQ ID NO: 1).
Variant human PARP2b: Insertion of 5'-gta tgc cag gaa ggt cat ggg
cca gca aaa ggg tct ctg-3' after nucleotide 204 (SEQ ID NO:1). This
extends the amino acid sequence by the insertion: GMPGRSWASKRVS
[0123] Nucleic acid derivatives also mean variants whose nucleotide
sequences in the region from -1 to -1000 in front of the start
codon have been modified so that gene expression and/or protein
expression is increased.
[0124] Besides the nucleotide sequence described above, the nucleic
acid constructs which can be used according to the invention
comprise in functional, operative linkage one or more other
regulatory sequences, such as promoters, amplification signals,
enhancers, polyadenylation sequences, origins of replication,
reporter genes, selectable marker genes and the like. This linkage
may, depending on the desired use, lead to an increase or decrease
in gene expression.
[0125] In addition to the novel regulatory sequences, it is
possible for the natural regulatory sequence still to be present in
front of the actual structural genes. This natural regulation can,
where appropriate, be switched off by genetic modification, and the
expression of the genes increased or decreased. However, the gene
construct may also have a simpler structure, that is to say no
additional regulatory signals are inserted in front of the
structural genes, and the natural promoter with its regulation is
not deleted. Instead, the natural regulatory sequence is mutated in
such a way that regulation no longer takes place, and gene
expression is enhanced or diminished. It is also possible to insert
additional advantageous regulatory elements at the 3' end of the
nucleic acid sequences. The nucleic acid sequences can be present
in one or more copies in the gene construct.
[0126] Advantageous regulatory sequences for the expression method
according to the invention are, for example, present in promoters
such as cos, tac, trp, tet, trp-tet, lpp, lac, lpp-lac, lacIq, T7,
T5, T3, gal, trc, ara, SP6, 1-PR or the 1-PL promoter, which are
advantageously used in Gram-negative bacteria. Other advantageous
regulatory sequences are present, for example, in the Gram-positive
promoters amy and SP02, in the yeast promoters ADC1, MFa, AC, P-60,
CYC1, GAPDH or in the plant promoters CaMV/355, SSU, OCS, lib4,
usp, STLS1, B33, nos or in the ubiquitin or phaseolin promoter.
[0127] It is possible in principle to use all natural promoters
with their regulatory sequences. It is also possible and
advantageous to use synthetic promoters.
[0128] Said regulatory sequences are intended to make specific
expression of the nucleic acid sequences and protein expression
possible. This may mean, for example, depending on the host
organism that the gene is expressed or overexpressed only after
induction, or that it is immediately expressed and/or
overexpressed.
[0129] The regulatory sequences or factors may moreover preferably
have a positive influence on, and thus increase or decrease, the
expression. Thus, enhancement of the regulatory elements may
advantageously take place at the level of transcription by using
strong transcription signals such as promoters and/or
enhancers.
[0130] However, it is also possible to enhance translation by, for
example, improving the stability of the mRNA.
[0131] Enhancers mean, for example, DNA sequences which bring about
increased expression via an improved interaction between RNA
polymerase and DNA.
[0132] The recombinant nucleic acid construct or gene construct is,
for expression in a suitable host organism, advantageously inserted
into a host-specific vector which makes optimal expression of the
genes in the host possible. Vectors are well known to the skilled
worker and are to be found, for example, in "Cloning Vectors"
(Pouwels P. H. et al., Ed., Elsevier, Amsterdam-New York-Oxford,
1985). Apart from plasmids, vectors also mean all other vectors
known to the skilled worker, such as, for example, phages, viruses,
such as SV40, CMV, baculovirus and adenovirus, transposons, IS
elements, phasmids, cosmids, and linear or circular DNA. These
vectors may undergo autonomous replication in the host organism or
chromosomal replication.
Expression of the Constructs:
[0133] The recombinant constructs according to the invention
described above are advantageously introduced into a suitable host
system and are expressed. Cloning and transfection methods familiar
to the skilled worker are preferably used in order to bring about
expression of said nucleic acids in the particular expression
system. Suitable systems are described, for example, in Current
Protocols in Molecular Biology, F. Ausubel et al., ed., Wiley
Interscience, New York 1997.
[0134] Suitable host organisms are in principle all organisms which
make it possible to express the nucleic acids according to the
invention, their allelic variants, their functional equivalents or
derivatives or the recombinant nucleic acid construct. Host
organisms mean, for example, bacteria, fungi, yeasts, plant or
animal cells. Preferred organisms are bacteria such as those of the
genera Escherichia, such as, for example, Escherichia coli,
Streptomyces, Bacillus or Pseudomonas, eukaryotic microorganisms
such as Saccharomyces cerevisiae, Aspergillus, higher eukaryotic
cells from animals or plants, for example Sf9 or CHO cells.
[0135] The gene product can also, if required, be expressed in
transgenic organisms such as transgenic animals such as, in
particular, mice, sheep, or transgenic plants. The transgenic
organisms may also be so-called knock-out animals or plants in
which the corresponding endogenous gene has been switched off, such
as, for example, by mutation or partial or complete deletion.
[0136] The combination of the host organisms and the vectors
appropriate for the organisms, such as plasmids, viruses or phages,
such as, for example, plasmids with the RNA polymerase/promoter
system, phages .lamda., .mu.. or other temperate phages or
transposons and/or other advantageous regulatory sequences forms an
expression system. The term expression systems preferably means,
for example, a combination of mammalian cells such as CHO cells,
and vectors, such as pcDNA3neo vector, which are suitable for
mammalian cells.
[0137] As described above, the gene product can also be expressed
advantageously in transgenic animals, e.g. mice, sheep, or
transgenic plants. It is likewise possible to program cell-free
translation systems with the RNA derived from the nucleic acid.
[0138] The gene product can also be expressed in the form of
therapeutically or diagnostically suitable fragments. To isolate
the recombinant protein it is possible and advantageous to use
vector systems or oligonucleotides which extend the cDNA by certain
nucleotide sequences and thus code for modified polypeptides which
serve to simplify purification. Suitable modifications of this type
are, for example, so-called tags which act as anchors, such as, for
example, the modification known as the hexa-histidine anchor, or
epitopes which can be recognized as antigens by antibodies
(described, for example, in Harlow, E. and Lane, D., 1988,
Antibodies: A Laboratory Manual. Cold Spring Harbor (N.Y.) Press).
These anchors can be used to attach the proteins to a solid support
such as, for example, a polymer matrix, which can, for example, be
packed into a chromatography column, or to a microtiter plate or to
another support.
[0139] These anchors can also at the same time be used to recognize
the proteins. It is also possible to use for recognition of the
proteins conventional markers such as fluorescent dyes, enzyme
markers which form a detectable reaction product after reaction
with a substrate, or radioactive markers, alone or in combination
with the anchors for derivatizing the proteins.
Production of Antibodies:
[0140] Anti-PARP2 antibodies are produced in a manner familiar to
the skilled worker. Antibodies mean both polyclonal, monoclonal,
human or humanized antibodies or fragments thereof, single chain
antibodies or also synthetic antibodies, likewise antibody
fragments such as Fv, Fab and F(ab').sub.2. Suitable production
methods are described, for example, in Campbell, A. M., Monoclonal
Anti-body Technology, (1987) Elsevier Verlag, Amsterdam, N.Y.,
Oxford and in Breitling, F. and Dubel, S., Rekombinante Antikorper
(1997), Spektrum Akademischer Verlag, Heidelberg.
Further Use of the Coding Sequence:
[0141] The present cDNA additionally provides the basis for cloning
the genomic sequence of the novel PARP genes. This also includes
the relevant regulatory or promoter sequence, which is available,
for example, by sequencing the region located 5' upstream of the
cDNA according to the invention or located in the intrans of the
genes. The cDNA sequence information is also the basis for
producing antisense molecules or ribozymes with the aid of known
methods (cf. Jones, J. T. and Sallenger, B. A. (1997) Nat.
Biotechnol. 15, 902; Nellen, W. and Lichtenstein, C. (1993) TIBS,
18, 419). The genomic DNA can likewise be used to produce the gene
constructs described above.
[0142] Another possibility of using the nucleotide sequence or
parts thereof is to generate transgenic animals. Transgenic
overexpression or genetic knock-out of the sequence information in
suitable animal models may provide further valuable information
about the (patho)physiology of the novel genes.
Therapeutic Applications:
[0143] In situations where there is a prevailing deficiency of a
protein according to the invention it is possible to employ several
methods for replacement. On the one hand, the protein, natural or
recombinant, can be administered directly or by gene therapy in the
form of its coding nucleic acid (DNA or RNA). It is possible to use
any suitable vectors for this, for example both viral and non-viral
vehicles. Suitable methods are described, for example, by Strauss
and Barranger in Concepts in Gene Therapy (1997), Walter de
Gruyter, publisher. Another alternative is provided by stimulation
of the endogenous gene by suitable agents.
[0144] It is also possible to block the turnover or the
inactivation of PARPs according to the invention, for example by
proteases. Finally, inhibitors or agonists of PARPs according to
the invention can be employed.
[0145] In situations where a PARP is present in excess or is
overactivated, various types of inhibitors can be employed. This
inhibition can be achieved both by antisense molecules, ribozymes,
oligonucleotides or antibodies, and by low molecular weight
compounds.
[0146] The active substances according to the invention, i.e. PARP
proteins, nucleic acids and PARP binding partners such as, for
example, antibodies or modulators, can be administered either as
single therapeutic active substances or as mixtures with other
therapeutic active substances. They can be administered as such,
but in general they are administered in the form of pharmaceutical
compositions, i.e. as mixtures of the active substance(s) with at
least one suitable pharmaceutical carrier or diluent. The active
substances or compositions can be administered in any way suitable
for the particular therapeutic purpose, e.g. orally or
parenterally.
[0147] The nature of the pharmaceutical composition and of the
pharmaceutical carrier or diluent depends on the required mode of
administration. Oral compositions can be, for example, in the form
of tablets or capsules and may contain customary excipients such as
binders (e.g. sirup, acacia, gelatin, sorbitol, tragacanth or
polyvinylpyrrolidone), bulking agents (e.g. lactose, sugar, corn
starch, calcium phosphate, sorbitol or glycine), lubricants (e.g.
magnesium stearate, talc, polyethylene glycol or silica),
disintegrants (e.g. starch) or wetting agents (e.g. sodium lauryl
sulfate). Oral liquid products may be in the form of aqueous or
oily suspensions, solutions, emulsions, sirups, elixirs or sprays
etc. or may be in the form of dry powders for reconstitution with
water or another suitable carrier. Liquid products of these types
may contain conventional additives, for example suspending agents,
flavorings, diluents or emulsifiers. It is possible to employ for
parenteral administration solutions or suspensions with
conventional pharmaceutical carriers. Parenteral administration of
active substances according to the invention advantageously takes
place using a liquid pharmaceutical composition which can be
administered parenterally, in particular intravenously. This
preferably contains an effective amount of at least one active
substance, preferably in dissolved form, in a pharmaceutically
acceptable carrier suitable for this purpose. Examples of
pharmaceutical carriers suitable for this purpose are, in
particular, aqueous solutions such as, for example, physiological
saline, phosphate-buffered saline, Ringer's solution, Ringer's
lactate solution and the like. The composition may moreover contain
further additions such as antioxidants, chelating agents or
antimicrobial agents.
[0148] The choice in each case of the dosage of the active
substances according to the invention and the particular dosage
schedule are subject to a decision of the treating physician. The
latter will select a suitable dose and an appropriate dosage
schedule depending on the chosen route of administration, on the
efficacy of the medicine in each case, on the nature and severity
of the disorder to be treated, and on the condition of the patient
and his response to the therapy. Thus, for example, the
pharmacologically active substances can be administered to a mammal
(human or animal) in doses of about 0.5 mg to about 100 mg per kg
of body weight and day. They can be administered in a single dose
or in several doses.
Nontherapeutic Applications:
[0149] The nucleic acids according to the invention, such as, for
example, cDNA, the genomic DNA, the promoter, and the polypeptide,
and partial fragments thereof, can also be used in recombinant or
nonrecombinant form for developing various test systems.
[0150] For example, it is possible to establish a test system which
is suitable for measuring the activity of the promoter or of the
protein in the presence of a test substance. The methods of
measurement in this case are preferably simple ones, e.g.
colorimetric, luminometric, fluorimetric, immunological or
radioactive, and allow preferably a large number of test substances
to be measured rapidly. Tests of this type are suitable and
advantageous for so-called high-throughput screening. These test
systems allow test substances to be assessed for their binding to
or their agonism, antagonism or inhibition of proteins according to
the invention.
[0151] Determination of the amount, activity and distribution of
the proteins according to the invention or their underlying mRNA in
the human body can be used for the diagnosis, for the determination
of the predisposition and for the monitoring of certain diseases.
Likewise, the sequence of the cDNA and the genomic sequence may
provide information about genetic causes of and predispositions to
certain diseases. It is possible to use for this purpose both
DNA/RNA probes and antibodies of a wide variety of types. The
nucleotide sequences according to the invention or parts thereof
can further be used in the form of suitable probes for detecting
point mutations, deletions or insertions.
[0152] The proteins according to the invention can further be used
to identify and isolate their natural ligands or interacting
partners. The proteins according to the invention can additionally
be used to identify and isolate artificial or synthetic ligands.
For this purpose, the recombinantly prepared or purified natural
protein can be derivatized in such a way that it has modifications
which permit linkage to support materials. Proteins bound in this
way can be incubated with various analytes, such as, for example,
protein extracts or peptide libraries or other sources of ligands.
Specifically bound peptides, proteins or low molecular weight,
non-proteinogenous substances can be isolated and characterized in
this way. Non-proteinogenous substances mean, for example, low
molecular weight chemical substances which may originate, for
example, from classical drug synthesis or from so-called substance
libraries which have been synthesized combinatorially.
[0153] The protein extracts used are derived, for example, from
homogenates of plants or parts of plants, microorganisms, human or
animal tissues or organs.
[0154] Ligands or interacting partners can also be identified by
methods like the yeast two-hybrid system (Fields, S. and Song, O.
(1989) Nature, 340, 245). The expression banks which can be
employed in this case may be derived, for example, from human
tissues such as, for example, brain, heart, kidney etc.
[0155] The nucleic acid sequences according to the invention and
the proteins encoded by them can be employed for developing
reagents, agonists and antagonists or inhibitors for the diagnosis
and therapy of chronic and acute diseases associated with the
expression or activation of one of the protein sequences according
to the invention, such as, for example, with increased or decreased
expression thereof. The reagents, agonists, antagonists or
inhibitors developed can subsequently be used to produce
pharmaceutical preparations for the treatment or diagnosis of
disorders. Examples of possible diseases in this connection are
those of the brain, of the peripheral nervous system, of the
cardiovascular system or of the eye, of septic shock, of rheumatoid
arthritis, diabetes, acute kidney failure, or of cancer.
[0156] The relevance of the proteins according to the invention for
said indications was verified using specific inhibitors in relevant
animal models.
[0157] The invention is now illustrated in detail with reference to
the following examples.
EXAMPLE 1
Isolation of the PARP2 and PARP3 cDNA
[0158] The present cDNA sequences were found for the first time on
sequence analysis of cDNA clones of a cDNA library from human brain
(Human Brain 5'Stretch Plus cDNA Library, # HL3002a, Clontech). The
mouse PARP3 clones were isolated from a "lambda triplex mouse brain
cDNA library" (Clontech order No. ML5004t). The sequences of these
clones are described in SEQ ID NO:1, 3, 7 and 9.
EXAMPLE 2
Expression of PARP2 and PARP3 in Human Tissues
[0159] The expression of human PARP2 and human PARP3 was
investigated in twelve different human tissues by Northern blot
analysis. A Human Multiple Tissue Northern Blot (MTNn) supplied by
Clontech (#7760-1 and #7780-1) was hybridized for this purpose with
an RNA probe. The probe was produced by in vitro transcription of
the corresponding cDNA of human PARP2 and human PARP3 in the
presence of digoxigenin-labeled nucleotides in accordance with the
manufacturer's method (BOEHRINGER MANNHEIM DIG Easy Hyb order No.
1603 558, DIG Easy Hyb method for RNA:RNA hybridization). The
protocol was modified to carry out the prehybridization: 2.times.1
h with addition of herring sperm DNA (10 mg/ml of hybridization
solution). Hybridization then took place overnight with addition of
herring sperm DNA (10 mg/ml of hybridization solution). The bands
were detected using the CDP-Star protocol (BOEHRINGER MANNHEIM
CDP-Star.TM. order No. 1685 627).
[0160] After stringent washing, the transcript of PARP2 was mainly
detected in human brain, heart, skeletal muscle, kidney and liver.
The transcript size of about 1.9 kb corresponds to the length of
the cDNA determined (1.85 kb) (cf. FIG. 2(A)).
[0161] In other tissues or organs, human PARP2 expression is
considerably weaker.
[0162] After stringent washing, the transcript of PARP3 was mainly
detected in heart, brain, kidney, skeletal muscle and liver.
Expression in other tissues (placenta, lung, pancreas) is
distinctly weaker (cf. FIG. 2(B)). There are at least 2 transcripts
for human PARP3, which can presumably be explained by different
polyadenylation sites or alternative splicing. Their size (about
2.2 kb and 2.5 kb respectively) corresponds to the length of the
cDNA determined (2.3 kb). Washing was carried out with
0.2.times.SSC/0.2% SDS at room temperature for 2.times.15 minutes
and then with 0.1.times.SSC/0.1% SDS at 65.degree. C. for
2.times.15 minutes (prepared from 20.times.SSC: 3M NaCl, 0.3M
sodium citrate, pH 7.0).
EXAMPLE 3
Production of Antibodies
[0163] Specific antibodies against the proteins according to the
invention were produced. These were used inter alia for analyzing
the tissue distribution at the protein level of PARP2 and PARP3 by
immunoblot (Western blot) analysis. Examples of the production of
such antibodies are indicated below.
[0164] The following peptides were prepared by synthesis in the
manner familiar to the skilled worker for the antibody production.
In some cases, a cysteine residue was attached to the N or C
terminals of the sequences in order to facilitate coupling to KLH
(keyhole limpet hemocyanin).
TABLE-US-00009 PARP-2: (amino acids 1-20; SEQ ID NO: 23)
NH.sub.2-MAARRRRSTGGGRARALNES-CO.sub.2H (amino acids 335-353; SEQ
ID NO: 24) NH.sub.2-KTELQSPEHPLDQHYRNLHC-CO.sub.2H PARP-3: (amino
acids 25-44 SEQ ID NO: 25) NH.sub.2-CKGRQAGREEDPFRSTAEALK-CO.sub.2H
(amino acids 230-248; SEQ ID NO: 26)
NH.sub.2-CKQQIARGFEALEALEEALK-CO.sub.2H
[0165] The production of an anti-PARP3 antibody is described as a
representative example.
[0166] For human PARP3, polyclonal antibodies were raised in
rabbits using a synthetic peptide having the peptide sequence
H.sub.2N-KQQIARG-FEALEALEEALK-CO.sub.2H (SEQ ID NO: 27) (amino
acids 230-248 of the human PARP3 protein sequence). The
corresponding mouse sequence differs in this region only by one
amino acid (H.sub.2N-KQQIARGFEALEALEEAMK-CO.sub.2H; SEQ ID NO: 28).
A cysteine was also attached to the N terminus in order to make it
possible for the protein to couple to KLH.
[0167] Rabbits were immunized a total of five times, at intervals
of 7-14 days, with the KLH-peptide conjugate. The antiserum
obtained was affinity-purified using the antigen. The specific IgG
fraction was isolated from the serum using the respective peptides
which, for this purpose, were initially immobilized on an affinity
column in the manner familiar to the skilled worker. The respective
antiserum was loaded onto this affinity column, and
non-specifically sorbed proteins were eluted with buffer. The
specifically bound IgG fraction was eluted with 0.2 M glycine/HCl
buffer pH 2.2. The pH was immediately increased using a 1M
TRIS/-HCl buffer pH 7.5. The eluate containing the IgG fraction was
mixed 1:1 (volume) with saturated ammonium sulfate solution and
incubated at +4.degree. C. for 30 min to complete the
precipitation. The resulting precipitate was centrifuged at 10,000
g and, after removal of the supernatant, dissolved in the minimum
amount of PBS/TBS. The resulting solution was then dialyzed against
PBS/TBS in the ratio 1:100 (volume). The antibodies were adjusted
to a concentration of about 100 .mu.g of IgG/ml. The PARP3
antibodies purified in this way had high specificity for PARP3.
Whereas mouse PARP3 was recognized well, there was no observable
cross-reaction with PARP 1 or PARP2.
EXAMPLE 4
Analysis of the Tissue Distribution by Immunoblot (Western
Blot)
[0168] The tissue distribution at the protein level was also
investigated for PARP2 and PARP3 by immunoblot (Western blot)
analysis.
Preparation of the Mouse Tissues for Protein Gels:
[0169] Tissues or cells were homogenized using a Potter or
Ultra-Turrax. For this, 0.5 g of tissue (or cells) was incubated in
5 ml of buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA, 6 mM MgCl.sub.2),
one tablet of protease inhibitor cocktail (Boehringer Mannheim,
order No. 1836153) and benzonase (purity grade I, MERCK) at
37.degree. C. for 30 min. Tissue samples from mice were produced
for heart, lung, liver, spleen, kidney, intestine, muscle, brain
and for human embryonic kidney cells (HEK293, human embryonal
kidney).
Protein Gels:
[0170] The NuPAGE system supplied by NOVEX was used according to
the instructions for protein gels. Polyacrylamide gels (NuPAGE
4-12% BisTris, NOVEX NP 0321), running buffer (MES-Running Buffer,
NOVEX NP 0002), antioxidant (NOVEX NP 0005), protein size standard
(Multi Mark Multi Colored Standard, NOVEX LC 5725), sample buffer
(NuPAGE LDS Sample Buffer (4.times.), NOVEX NP 0007) were used. The
gels were run for 45 minutes at a voltage of 200 V.
Western Blot:
[0171] Western blots were carried out using the NOVEX system in
accordance with instructions. A nitrocellulose membrane
(Nitrocellulose Pore size 45 .mu.m, NOVEX LC 2001) was used. The
transfer took 1 hour at a current of 200 mA. The transfer buffer
consisted of 50 ml of transfer buffer concentrate (NOVEX NP 0006),
1 ml of antioxidant (NOVEX NP 0002), 100 ml of analytical grade
methanol and 849 ml of double-distilled water.
[0172] Besides the blots produced in this way, also used were
premade blots, for example from Chemicon (mouse brain blot,
Chemicon, catalog No.: NS 106 with the tissues 1. frontal cortex,
2. posterior cortex, 3. cerebellum, 4. hippocampus, 5. olfactory
bulb, 6. striatum, 7. thalamus, 8. mid brain, 9. entorhinal cortex,
10. pons, 11. medulla, 12. spinal cord).
Antibody Reaction with PARP3:
[0173] The Western blots were blocked in TBST (TBS+0.3% Tween 20)
with 5% dry milk powder for at least 2 hours (TBS: 100 mM Tris pH
7.5, 200 mM NaCl). The antibody reaction with the primary anti-body
(dilution 1:1000) took place in TBST with 5% dry milk powder (see
above) at room temperature for at least 2 hours or at 4.degree. C.
overnight, with gentle agitation (vertical rotator). This was
followed by washing three times in TBST for 5 minutes. Incubation
with the secondary antibody (anti-rabbit IgG, peroxidase-coupled,
SIGMA A-6154, dilution 1:2000) took place in TBST with 5% dry milk
powder for 1 hour. This was followed by washing three times for 5
minutes each time as above. The subsequent detection was based on
chemiluminescence using the SUPER BLAZE kit (Pierce, Signal BLAZE
Chemiluminescent Substrate 34095) as stated by the manufacturer.
The "Lumi-Film" (Chemiluminescent Detection Film, Boehringer order
No: 1666916) was used. The films were developed for about 2 min
(X-ray developer concentrate, ADEFO-Chemie GmbH), hydrated, fixed
for about 4 min (Acidofix 85 g/l/AGFA), hydrated and then
dried.
EXAMPLE 5
Preparation of the Enzymes
[0174] For comparison, human PARP1 was expressed recombinantly in
the baculovirus system in the manner familiar to the skilled worker
and partially purified as described (Shah et al., Analytical
Biochemistry 1995, 227, 1-13). Bovine PARP1 in a purity of 30-50%
(c=0.22 mg/ml, spec. activity 170 nmol of ADP-ribose/min/mg of
total protein at 25.degree. C.) was purchased from BIOMOL (order
No. SE-165). Human and mouse PARP2 and PARP3 were expressed
recombinantly in the baculovirus system (Bac-to-Bac system, BRL
LifeScience). For this purpose, the appropriate cDNAs were cloned
to the pFASTBAC-1 vector. Preparation of recombinant baculovirus
DNA by recombination in E. coli was followed by transfection of
insect cells (Sf9 or High-Five) with the appropriate recombinant
baculovirus DNAs. Expression of the corresponding proteins was
verified by Western blot analysis. Virus strains were amplified in
the manner familiar to the skilled worker. Larger amounts of
recombinant proteins were obtained by infecting 500 ml of insect
cell culture (2.times.10.sup.6 cells/ml) with viruses in an MOI
(multiplicity of infection; ratio of viruses to cells) of 5-10 and
incubating for 3 to 4 days. The insect cells were then pelleted by
centrifugation, and the proteins were purified from the pellet.
[0175] The purification took place by classical methods of protein
purification familiar to the skilled worker, detecting the enzymes
with appropriate specific antibodies. In some cases, the proteins
were also affinity-purified on a 3-aminobenzamide affinity column
as described (Burtscher et al., Anal Biochem 1986, 152:285-290).
The purity was >90%.
EXAMPLE 6
Assay Systems for Determining the Activity of PARP2 and PARP3 and
the Inhibitory Action of Effectors on PARP1, PARP2 and PARP3
a) Production of Antibodies Against Poly(ADP-Ribose)
[0176] It is possible to use poly(ADP-ribose) as antigen for
generating anti-poly(ADP-ribose) antibodies. The production of
anti-poly(ADP-ribose) antibodies is described in the literature
(Kanai Y et al. (1974) Biochem Biophys Res Comm 59:1, 300-306;
Kawamaitsu H et al. (1984) Biochemistry 23, 3771-3777; Kanai Y et
al. (1978) Immunology 34, 501-508).
[0177] The following were used, inter alia: anti-poly(ADP-ribose)
anti-bodies (polyclonal antiserum, rabbits), BIOMOL; order No.
SA-276, anti-poly(ADP-ribose) antibodies (monoclonal, mouse; clone
lOH; hybridoma supernatant, affinity-purified).
[0178] The antisera or monoclonal antibodies obtained from
hybridoma supernatant were purified by protein A affinity
chromatography in the manner familiar to the skilled worker.
b) ELISA
Materials:
ELISA Color Reagent: TMB Mix, SIGMA T-8540
[0179] A 96-well microtiter plate (FALCON Micro-Test III.TM.
Flexible Assay Plate, #3912) was coated with histones (SIGMA,
H-7755). Histones were for this purpose dissolved in carbonate
buffer (0.05M Na.sub.2HCO.sub.3; pH 9.4) in a concentration of 50
.mu.g/ml. The individual wells of the microtiter plate were each
incubated with 150 .mu.l of this histone solution at room
temperature for at least 2 hours or at 4.degree. C. overnight. The
wells are then blocked by adding 150 .mu.l of a 1% BSA solution
(SIGMA, A-7888) in carbonate buffer at room temperature for 2
hours. This is followed by three washing steps with washing buffer
(0.05% Tween10 in 1.times.PBS; PBS (Phosphate buffered saline;
Gibco, order No. 10010): 0.21 g/l KH.sub.2PO.sub.4, 9 g/l NaCl,
0.726 g/l Na.sub.2HPO.sub.4.7H.sub.2O, pH 7.4). Washing steps were
all carried out in a microtiter plate washer ("Columbus" microtiter
plate washer, SLT-Labinstruments, Austria).
[0180] Required for the enzyme reaction were an enzyme reaction
solution and a substrate solution, in each case as a premix. The
absolute amount of these solutions depended on the intended number
of assay wells.
Composition of the Enzyme Reaction Solution Per Well:
[0181] 4 .mu.l of PARP reaction buffer (1M Tris-HCl pH 8.0, 100 mM
MgCl.sub.2, 10 mM DTT) [0182] 20 ng of PARP1 (human or bovine) or 8
ng PARP2 (human or mouse) [0183] 4 .mu.l of activated DNA (1 mg/ml;
SIGMA, D-4522) [0184] H.sub.2O ad 40 .mu.l
Composition of the Substrate Solution Per Well:
[0184] [0185] 5 .mu.l of PARP reaction buffer (10.times.) [0186]
0.8 .mu.l of NAD solution (10 mM, SIGMA N-1511) [0187] 44 .mu.l
H.sub.2O
[0188] Inhibitors were dissolved in 1.times.PARP reaction buffer.
DMSO, which was occasionally used to dissolve inhibitors in higher
concentrations, was no problem up to a final concentration of 2%.
For the enzyme reaction, 40 .mu.l of the enzyme reaction solution
were introduced into each well and incubated with 10 .mu.l of
inhibitor solution for 10 minutes. The enzyme reaction was then
started by adding 50 .mu.l of substrate solution per well. The
reaction was carried out at room temperature for 30 minutes and
then stopped by washing three times with washing buffer.
[0189] The primary antibodies employed were specific
anti-poly(ADP-ribose) antibodies in a dilution of 1:5000. Dilution
took place in antibody buffer (1% BSA in PBS; 0.05% Tween20). The
incubation time for the primary antibodies was one hour at room
temperature. After subsequently washing three times with washing
buffer, incubation was carried out with the secondary antibody
(anti-mouse IgG, Fab fragments, peroxidase-coupled, Boehringer
Mannheim, order No. 1500.686; anti-rabbit IgG, peroxidase-coupled,
SIGMA, order No. A-6154) in a dilution of 1:10,000 in antibody
buffer at room temperature for one hour. Washing three times with
washing buffer was followed by the color reaction using 100 .mu.l
of color reagent (TMB mix, SIGMA) per well at room temperature for
about 15 min. The color reaction was stopped by adding 100 .mu.l of
2M H.sub.2SO.sub.4. This was followed by immediate measurement in
an ELISA plate reader (EAR340AT "Easy Reader", SLT-Labinstruments,
Austria) (450 nm versus 620 nm). The measurement principle is
depicted diagrammatically in FIG. 6.
[0190] Various concentrations were used to construct a dose-effect
plot to determine the K.sub.i value of an inhibitor. Values are
obtained in triplicate for a particular inhibitor concentration.
Arithmetic means are determined using Microsoft.COPYRGT. Excel. The
IC.sub.50 is determined using the Microcal.COPYRGT. Origin Software
(Vers. 5.0) ("Sigmoidal Fit"). Conversion of the IC.sub.50 value is
calculated in this way into K.sub.i values took place by using
"calibration inhibitors". The "calibration inhibitors" were also
measured in each analysis. The K.sub.i values of the "calibration
inhibitors" were determined in the same assay system by analysis of
the Dixon diagram in the manner familiar to the skilled worker.
b) HTRF (Homogenous Time-Resolved Fluorescence) Assay
[0191] In the HTRF PARP assay according to the invention, histones,
as target proteins for modification by PARP, are labeled indirectly
with an XL665 fluorophore. The anti poly(ADP ribose) antibody is
directly labeled with a europium cryptate (anti-PAR-cryptate). If
the XL665 fluorophore is in the direct vicinity in space, which is
ensured by binding to the poly(ADP-ribose) on the histone, then
energy transfer is possible. The emission at 665 nm is thus
directly proportional to the amount of bound antibody, which in
turn is equivalent to the amount of poly(ADP-ribose). The measured
signal thus corresponds to the PARP activity. The measurement
principle is depicted diagrammatically in FIG. 7. The materials
used are identical to those used in the ELISA (see above) unless
expressly indicated.
[0192] Histones were dissolved in a concentration of 3 mg/ml in
Hepes buffer (50 mM, pH=7.5). Biotinylation took place with
sulfa-NHS-LC-biotin (Pierce, #21335T). A molar ratio of 4 biotin
molecules per histone was used. The incubation time was 90 minutes
(RT). The biotinylated histones were then purified on a G25 SF
HR10/10 column (Pharmacia, 17-0591-01) in Hepes buffer (50 mM,
pH=7.0) in order to remove excess biotinylation reagent. The
anti-poly(ADP-ribose) antibody was labeled with europium cryptate
using bifunctional coupling reagents (Lopez, E. et al., Clin. Chem.
39(2), 196-201 (1993); U.S. Pat. No. 5,534,622).
[0193] Purification took place on a G25SF HR10/30 column. A molar
ratio of 3.1 cryptates per antibody was achieved. The yield was
25%. The conjugates were stored at -80.degree. C. in the presence
of 0.1% BSA in phosphate buffer (0.1M, pH=7).
For the Enzyme Reaction, the Following were Pipetted into Each
Well: [0194] 10 .mu.l of PARP solution in PARP HTRF reaction buffer
(50 mM Tris-HCl pH 8.0, 10 mM MgCl.sub.2, 1 mM DTT) with 20 ng of
PARP1 (human or bovine) or 8 ng of PARP2 (human or mouse) [0195] 10
.mu.l of activated DNA in PARP HTRF reaction buffer (50 .mu.g/ml)
[0196] 10 .mu.l of biotinylated histones in PARP HTRF reaction
buffer (1.25 .mu.M) [0197] 10 .mu.l of inhibitor in PARP HTRF
reaction buffer
[0198] These reagents were incubated for 2 minutes before the
reaction was started by adding [0199] 10 .mu.l of NAD solution in
PARP HTRF reaction buffer (41 .mu.M/ml). The reaction time was 30
minutes at room temperature.
[0200] The reaction was then stopped by adding [0201] 10 .mu.l of
PARP inhibitor (25 .mu.M, K.sub.i=10 nM) in "Revelation" buffer
(100 mM Tris-HCl pH 7.2, 0.2 M KF, 0.05% BSA).
[0202] The following were then added: [0203] 10 .mu.l of EDTA
solution (SIGMA, E-7889, 0.5 M in H.sub.2O) [0204] 100 .mu.l of
Sa-XL665 (Packard Instruments) in "Revelation" buffer (15-31.25 nM)
[0205] 50 .mu.l of anti-PAR cryptate in "Revelation" buffer
(1.6-3.3 nM).
[0206] Measurement was then possible after 30 minutes (up to 4
hours). The measurement took place in a "discovery HTRF microplate
analyzer" (Canberra Packard Instruments). The K.sub.i values were
calculated as described for the ELISA.
EXAMPLE 7
Test Systems for Determining the Therapeutic Efficacy of PARP
Inhibitors Novel PARP Inhibitors can have their Therapeutic
Efficacy Checked in Relevant Pharmacological Models. Examples of
Some Suitable Models are Listed in Table 1
TABLE-US-00010 [0207] Disorder Model Literature Neurodegenerative
disorders INMDA excitotoxicity in mice (stroke, Parkinson's, etc.)
or rats See below for description Stroke Permanent MCAO ("middle
Tokime, T. et al t cerebral arterial occlusion") J. Cereb. Blood
Flow nvletab., 18 (9): 991-7, 1998. Guegan, C., Brain Research.
Molecular Brain Research, 55 (1): 133-40, 1998. Transient, focal
MCAO in rats Eliasson MJL et al., Nat Med or mice 1997, 3:
1089-1095. Endres, M et al., J Cereb Blood Flow Metab 1997, 17:
1143-1151. Takahashi K et al., J Cereb Blood Flow Metab 1997, 17:
1137-1142. Parkinson's disease MPTP (1-methyl-4-phenyl- Cosi C, et
al./Brain Res., 1998 1,2,3,6-tetrahydropyridine)toxicity 809 (1):
58-67. in mice/rats Cosi C, et al., Brain Res., 1996 729 (2):
264-9. Myocardial infarct Coronary vessel occlusion in Richard V,
et al., Br. J. rats, pigs or rabbits Pharmacol 1994, 113, 869-876.
Thiemermann C, et Al., Proc Natl Acad Sci USA. 1997, 94 (2):
679-83. Zingarelli B, et al., Cardiovasc Res. 1997, 36(2): 205-15.
Langendorf heart model in rats or rabbits See below for description
Septic shock Endotoxin shock in rats Szabo C, et al., J Clin
Invest, 1997, 100(3): 723-35. Zymosan- or carrageenan- Szabo C, et
al. J induced multiple organ failure Exp Med. 1997, 186(7): 1041-9.
in rats or mice Cuzzocrea S, et al. Eur J Pharmacol. 1998, 342 (1):
67-76. Rheumatoid arthritis Adjuvant- or collagen-induced Szabo C,
et al., Proc Natl Acad arthritis in rats or mice Sci USA. 1998,
95(7): 3867-72. Diabetes Streptozotocin- and alloxan- Uchgata Y et
al., induced or obesity-associated Diabetes 1983, 32: 316-318.
Masiello P et al., Diabetologia 1985, 28: 683-686. Shimabukuro M et
al., J Clin Invest 1997, 100: 290-295. Cancer In vitro model; see
below Schlicker et al., 1999, 75 (1), 91-100.
a) NMDA Excitotoxicity Model
[0208] Glutamate is the most important excitory neurotransmitter in
the brain. Under normal conditions, glutamate is secreted into the
synaptic cleft and stimulates the post-synaptic glutamate
receptors, specifically the glutamate receptors of the "NMDA" and
"AMPA" types. This stimulation plays a significant part in numerous
functions of the brain, including learning, memory and motor
control.
[0209] Under the conditions of acute and chronic neurodegeneration
(e.g. stroke), however, there is a great increase in the
presynaptic glutamate secretion, resulting in excessive stimulation
of the receptors. This leads to death of the cells stimulated in
this way. These increased glutamate activities occur in a number of
neurological disorders or psychological disturbances and lead to
states of overexcitation or toxic effects in the central nervous
system (CNS) but also in the peripheral nervous system. Thus,
glutamate is involved in a large number of neurodegenerative
disorders, in particular neurotoxic disturbances following hypoxia,
anoxia, ischemia and after lesions like those occurring after
stroke and trauma, and stroke, Alzheimer's disease, Huntington's
disease, amyotrophic lateral sclerosis (ALS; "Lou Gehring's
disease"), cranial trauma, spinal cord trauma, peripheral neuro
pathies, AIDS dementia and Parkinson's disease. Another disease in
which glutamate receptors are important is epilepsy (cf. Brain Res
Bull 1998; 46(4):281-309, Eur Neuropsychopharmacol 1998, 8(2):
141-52.).
[0210] Glutamate effects are mediated through various receptors.
One of these receptors is called the NMDA (N-methyl-D-aspartate)
receptor after a specific agonist (Arzneim.Forschung 1990, 40,
511-514; TIPS, 1990, 11, 334-338; Drugs of the Future 1989, 14,
1059-1071). N-Methyl-D-aspartate is a strong agonist of a
particular class of glutamate receptors ("NMDA" type). Stimulation
of the NMDA receptor leads to influx of calcium into the cell and
the generation of free radicals. The free radicals lead to DNA
damage and activation of PARP. PARP in turn causes cell death
through depletion of high-energy phosphates (NAD and ATP) in the
cell. This explains the toxicity of NMDA. Treatment of animals with
NMDA can therefore be regarded as a model of the abovementioned
disorders in which excitotoxicity is involved.
[0211] Because of the importance of glutamate receptors in
neurodegeneration, many pharmacological approaches to date have
been directed at specific blocking of precisely these receptors.
However, because of their importance in normal stimulus conduction,
these approaches have proved to be problematic (side effects). In
addition, stimulation of the receptors is an event which takes
place very rapidly so that administration of the receptors often
comes too late ("time window" problem). Thus there is a great need
for novel principles of action and inhibitors of NMDA-related
neurotoxicity.
[0212] Protection against cerebral overexcitation by excitatory
amino acids (NMDA antagonism in mice) can be regarded as adequate
proof of the activity of a pharmacological effector of PARP in
disorders based on excitotoxicity. Intracerebral administration of
excitatory amino acids (EAA) induces such massive overexcitation
that it leads within a short time to convulsions and death of the
animals (mice).
[0213] In the present case there was unilateral
intracerebroventricular administration of 10 .mu.l of a 0.035%
strength aqueous NMDA solution 120 minutes after intraperitoneal
(i.p.) administration of the test substance. These symptoms can be
inhibited by systemic, e.g. intraperitoneal, administration of
centrally acting drugs. Since excessive activation of EAA receptors
in the central nervous system plays an important part in the
pathogenesis of various neurological disorders, information can be
gained from the detected EAA antagonism in vivo about possible
therapeutic utilizability of the substances for such CNS disorders.
An ED50 at which 50% of the animals are, due to preceding i.p.
administration of the measured substance, free of symptoms with a
fixed dose of NMDA was determined as a measure of the activity of
the substances.
b) Langendorf Heart Model (Model for Myocardial Infarct)
[0214] Male Sprague-Dawley rats (bodyweight 300-400 g; origin
Janvier, Le Genest-St-Isle, France) were used for the test. The
rats were treated orally by gavage with the active substance or
placebo (volume: 5 ml/kg). 50 minutes later, heparin is
administered intraperitoneally (Liquemin N Roche, 125 IU/animal in
0.5 ml). The animals are anesthesized with Inactin.RTM. T133
(thiobetabarbital sodium 10%), fixed on the operating table,
tracheotomized and ventilated with a "Harvard ventilatory pump" (40
beats/min, 4.5 ml/beat). Thoracotomy was followed by immediate
catheterization of the aorta, removal of the heart and immediate
retrograde perfusion. The hearts were perfused with a constant
pressure of 75 mmHg, which is achieved using a "Gilson Miniplus 2
perfusion pump". Composition of the perfusate (mmol/1): NaCl 118,
KCl 4.7, CaCl.sub.2.times.2 H.sub.2O 2.52, MgSO.sub.4.times.7
H.sub.2O 1.64, NaHCO.sub.3 24.88, KH.sub.2PO.sub.4 1.18, glucose
11. The temperature is kept at 37.degree. C. throughout the
experiment. Functional parameters were continuously recorded using
a "Gould 4-channel recorder". Measurements were made of the
left-ventricular pressure (LVP; mmHg), LVEDP (mmHg), enzyme release
(creatine kinase, mU/ml/g), coronary flow rate (ml/min), HR (pulse
rate, min.sup.-1). The left-ventricular pressure was measured using
a liquid-filled latex balloon and a Statham23 Db pressure
transducer. The volume of the balloon was initially adjusted to
reach an LVEDP (left-ventricular end-diastolic pressure) of about
12 mmHg. The dP/dt.sub.max (maximum pumping force) is derived from
the pressure signal using a differentiator module. The heart rate
was calculated from the pressure signal. The flow rate was
determined using a drop counter (BMT Messtechnik GmbH Berlin).
After an equilibration time of 20 minutes, the hearts were
subjected to a 30-minute global ischemia by stopping the perfusate
supply while keeping the temperature at 37.degree. C. During the
following 60-minute reperfusion period, samples of the perfusate
were taken after 3, 5, 10, 15, 30, 45 and 60 min for analysis of
creatine kinase (CK) activity. Means and standard deviations for
the measured parameters were analyzed statistically (Dunnett test).
The significance limit was p=0.05.
[0215] The experiment on rabbit hearts was carried out similarly.
Male white New Zealand rabbits (obtained from: Interfauna) were
used. The hearts were prepared as described above for the rat
model. The perfusion pressure was set at a maximum of 60 mmHg and
the flow rate at about 25 ml/min. The equilibration time was about
30 min. The substance was administered by infusion directly
upstream of the heart. 15 min after starting the infusion, a
30-minute global ischemia was caused by stopping the flow while
maintaining the temperature of the heart. A 30-minute reperfusion
followed. Perfusate was taken for investigation of CK activity
before administration of the substance, after 15 min and at various
times (5, 10, 15, 20, 30 min) during the reperfusion. The following
parameters were measured: LVP (mmHg), LVEDP, LVdP/dt, PP (mmHg), HR
(pulse rate; beats/min), CK activity (U/min/g heart weight).
c) Animal Model for Acute Kidney Failure
[0216] The protective effect of intravenous administration of PARP
inhibitors (4 days) on the kidney function of rats with
postischemic acute kidney failure was investigated.
[0217] Male Sprague-Dawley rats (about 330 g at the start of the
experiments; breeder: Charles River) were used. 10-15 animals were
employed per experimental group. Administration of active
substance/placebo took place continuously with an osmotic micropump
into the femoral vein. Orbital blood was taken (1.5 ml of whole
blood) under inhalation anesthesia with enflurane (Ethrane Abbot,
Wiesbaden).
[0218] After the initial measurements (blood sample) and
determination of the amount of urine excreted in 24 h, the rats
were anesthetized ("Nembutal", pentobarbital sodium, Sanofi CEVA;
50 mg/kg i.p., volume injected 1.0 ml/kg) and fastened on a
heatable operating table (37.degree. C.). 125 IU/kg heparin
(Liquemin N, Roche) were administered i.v. into the caudal vein.
The abdominal cavity was opened and the right kidney was exposed.
The branching-off renal artery was exposed and clamped off
superiorly using bulldog clamps (Diefenbach 38 mm) The left renal
artery was likewise exposed and clamped off (superiorly, about half
way to the kidney). During the operation, an osmotic micropump was
implanted into the femoral vein. The intestine was reinserted and
the fluid loss was compensated with luke-warm 0.9% NaCl. The
animals were covered with a moist cloth and kept warm under red
light. After 40 min, the appearance of the kidneys was recorded,
and the clamps were removed, first the right then the left. The
intestine was put back and 2 drops of antibiotic (Tardomyocel,
Bayer) were added. The abdominal wall was closed with sterile cat
gut (Ethicon No. 4) and treated once more with 1 drop of
antibiotic. The epidermis was sutured with sterile Ethibond Exel
(Ethicon) No. 3/0, and the suture was sprayed with Nebacetin N
(Yamanouchi) wound spray. A tenth of a daily dose of drug/placebo
is given as i.v. bolus.
[0219] Samples and blood were taken for investigating biochemical
parameters in the serum and urine: Na, K, creatinine, protein (only
in urine), on days 1, 2 and 4 of the experiment. In addition, the
feed and water consumption, bodyweight and urine volume were
recorded. After 14 days, the animals were sacrificed and the
kidneys were assessed.
[0220] The assessment excluded all animals which died of an infarct
during the experiment or showed an infarct at necropsy on day 14.
The creatinine clearance and the fractional sodium excretion were
calculated as kidney function parameters, comparing treated animals
with control and sham.
d) In Vitro Model for Radiosensitization (Tumor Therapy)
[0221] MCF-7-cells (human breast carcinoma) were cultivated in
Dulbecco's modified Eagle's medium with 10% heat-inactivated FCS
and 2 mM L-glutamine Cells were seeded out overnight in cell
densities of 100, 1000 or 10,000 cells per well in a 6-well plate
and then exposed to ionizing radiation with a dose in the range
from 0 to 10 Gy (.sup.137Cs, Shepard Mark, model I-68A, dose rate
3.28 Gy/min) 10 days after the irradiation, the experiment was
assessed, counting colonies with fifty cells as positive.
e) Stroke Model (Focal Cerebral Ischemia; MCA (Middle Cerebral
Artery) Occlusion on a Rat)
[0222] A focal ischemia was performed by means of cauterisation of
the right distal MCA on Sprague-Dawley or Long-Evans rats. The rats
may be treated before or after the beginning of the MCA occlusion
with modulators of the proteins of the invention. As a rule, doses
of 1-10 mg/kg are chosen (bolus application), optionally followed
by a continuous infusion of 0.5-5 mg/kg/h.
[0223] The rats are anesthetised with halothane in a mixture of 70%
nitrogen and 30% oxygen (4% at initial phase and 0.8-1.2% during
the operation). The body temperature was permanently measured
rectally and was kept constant at 37.5.degree. C..+-.0.5.degree. C.
by means of a controllable heating blanket. Moreover, arterial
blood pressure, arterial pH, (Pa(O.sub.2) and Pa(CO.sub.2) were
optionally measured by means of a tail vein catheder. Thereafter,
the focal iscehmia was carried out using the method of Chen et al.
(Stroke 17: 738-743; 1986) or Liu et al. (Am. J. Physiol. 256:
H589-593; 1989) by means of continuous cauterisation of the distal
part of the right MCA. When the operation was terminated, the
animals were kept in a warm environment for a further 24 hours.
Then they were killed with the use of CO.sub.2 and decapitated.
Their brains were taken, shock-frozen (dry ice or liquid nitrogen)
and stored at -80.degree. C. The brains were cut into 0.02 mm thick
slices and every 20th cut was used for the subsequent analysis. The
corresponding cuts are stained with cresyl violet (Nissl staining)
Alternatively, TTC (2,3,4-triphenyltetrazoliumchloride) may be used
for staining. The infarct volume may then be analysed under a
microscope. For exact quantification, a computer-based image
analyzing software may be used (J. Cereb. Clood Flow Metabol. 10:
290-293; 1990).
f) Septic Shock
[0224] Groups of 10 male C57/BL mice (body weight 18-20 g) are
treated with LPS (lipopolysaccharide, from E. coli, LD.sub.100 20
mg/animal i. v.) plus galactosamine (20 mg/animal i. v.). the
substance to be tested is applied i. p. or i. v. during three
succeeding days (e. g. 1-10 mg/kg), with the first dose being
administered 30 minutes after the LPS treatment. The death rate is
determined every 12 hours. Alternatively, the substance may also be
applied in several doses spread over the days.
g) Determination of Altered Gene Expression in Aging Cells
[0225] The aging of cells is simulated by changing the cell culture
media from the complete medium with a reduced serum concentration
and thereafter is analysed by means of quantitative PCR or Northern
Blotting (Linskens et al., Nucleic Acids Res. 1995, 23(16):
3244-51). As typical markers for the aging of the skin for example
collagen or elastin may be used. Human fibroblasts or fibroblast
cell lines are used which simulate the aging of the skin.
Modulators of the proteins of the invention are added to the medium
and their effect on the changing of the gene expression is
observed. An increased production of elastin in cells with a
reduced aging process caused by means of said modulators may be
observed.
Sequence CWU 1
1
4011843DNAHomo sapiensCDS(3)..(1715)product is Poly ADP Ribose
Polymerase; from brain tissue 1cc atg gcg gcg cgg cgg cga cgg agc
acc ggc ggc ggc agg gcg aga 47 Met Ala Ala Arg Arg Arg Arg Ser Thr
Gly Gly Gly Arg Ala Arg 1 5 10 15 gca tta aat gaa agc aaa aga gtt
aat aat ggc aac acg gct cca gaa 95Ala Leu Asn Glu Ser Lys Arg Val
Asn Asn Gly Asn Thr Ala Pro Glu 20 25 30 gac tct tcc cct gcc aag
aaa act cgt aga tgc cag aga cag gag tcg 143Asp Ser Ser Pro Ala Lys
Lys Thr Arg Arg Cys Gln Arg Gln Glu Ser 35 40 45 aaa aag atg cct
gtg gct gga gga aaa gct aat aag gac agg aca gaa 191Lys Lys Met Pro
Val Ala Gly Gly Lys Ala Asn Lys Asp Arg Thr Glu 50 55 60 gac aag
caa gat gaa tct gtg aag gcc ttg ctg tta aag ggc aaa gct 239Asp Lys
Gln Asp Glu Ser Val Lys Ala Leu Leu Leu Lys Gly Lys Ala 65 70 75
cct gtg gac cca gag tgt aca gcc aag gtg ggg aag gct cat gtg tat
287Pro Val Asp Pro Glu Cys Thr Ala Lys Val Gly Lys Ala His Val Tyr
80 85 90 95 tgt gaa gga aat gat gtc tat gat gtc atg cta aat cag acc
aat ctc 335Cys Glu Gly Asn Asp Val Tyr Asp Val Met Leu Asn Gln Thr
Asn Leu 100 105 110 cag ttc aac aac aac aag tac tat ctg att cag cta
tta gaa gat gat 383Gln Phe Asn Asn Asn Lys Tyr Tyr Leu Ile Gln Leu
Leu Glu Asp Asp 115 120 125 gcc cag agg aac ttc agt gtt tgg atg aga
tgg ggc cga gtt ggg aaa 431Ala Gln Arg Asn Phe Ser Val Trp Met Arg
Trp Gly Arg Val Gly Lys 130 135 140 atg gga cag cac agc ctg gtg gct
tgt tca ggc aat ctc aac aag gcc 479Met Gly Gln His Ser Leu Val Ala
Cys Ser Gly Asn Leu Asn Lys Ala 145 150 155 aag gaa atc ttt cag aag
aaa ttc ctt gac aaa acg aaa aac aat tgg 527Lys Glu Ile Phe Gln Lys
Lys Phe Leu Asp Lys Thr Lys Asn Asn Trp 160 165 170 175 gaa gat cga
gaa aag ttt gag aag gtg cct gga aaa tat gat atg cta 575Glu Asp Arg
Glu Lys Phe Glu Lys Val Pro Gly Lys Tyr Asp Met Leu 180 185 190 cag
atg gac tat gcc acc aat act cag gat gaa gag gaa aca aag aaa 623Gln
Met Asp Tyr Ala Thr Asn Thr Gln Asp Glu Glu Glu Thr Lys Lys 195 200
205 gag gaa tct ctt aaa tct ccc ttg aag cca gag tca cag cta gat ctt
671Glu Glu Ser Leu Lys Ser Pro Leu Lys Pro Glu Ser Gln Leu Asp Leu
210 215 220 cgg gta cag gag tta ata aag ttg atc tgt aat gtt cag gcc
atg gaa 719Arg Val Gln Glu Leu Ile Lys Leu Ile Cys Asn Val Gln Ala
Met Glu 225 230 235 gaa atg atg atg gaa atg aag tat aat acc aag aaa
gcc cca ctt ggg 767Glu Met Met Met Glu Met Lys Tyr Asn Thr Lys Lys
Ala Pro Leu Gly 240 245 250 255 aag ctg aca gtg gca caa atc aag gca
ggt tac cag tct ctt aag aag 815Lys Leu Thr Val Ala Gln Ile Lys Ala
Gly Tyr Gln Ser Leu Lys Lys 260 265 270 att gag gat tgt att cgg gct
ggc cag cat gga cga gct ctc atg gaa 863Ile Glu Asp Cys Ile Arg Ala
Gly Gln His Gly Arg Ala Leu Met Glu 275 280 285 gca tgc aat gaa ttc
tac acc agg att ccg cat gac ttt gga ctc cgt 911Ala Cys Asn Glu Phe
Tyr Thr Arg Ile Pro His Asp Phe Gly Leu Arg 290 295 300 act cct cca
cta atc cgg aca cag aag gaa ctg tca gaa aaa ata caa 959Thr Pro Pro
Leu Ile Arg Thr Gln Lys Glu Leu Ser Glu Lys Ile Gln 305 310 315 tta
cta gag gct ttg gga gac att gaa att gct att aag ctg gtg aaa 1007Leu
Leu Glu Ala Leu Gly Asp Ile Glu Ile Ala Ile Lys Leu Val Lys 320 325
330 335 aca gag cta caa agc cca gaa cac cca ttg gac caa cac tat aga
aac 1055Thr Glu Leu Gln Ser Pro Glu His Pro Leu Asp Gln His Tyr Arg
Asn 340 345 350 cta cat tgt gcc ttg cgc ccc ctt gac cat gaa agt tac
gag ttc aaa 1103Leu His Cys Ala Leu Arg Pro Leu Asp His Glu Ser Tyr
Glu Phe Lys 355 360 365 gtg att tcc cag tac cta caa tct acc cat gct
ccc aca cac agc gac 1151Val Ile Ser Gln Tyr Leu Gln Ser Thr His Ala
Pro Thr His Ser Asp 370 375 380 tat acc atg acc ttg ctg gat ttg ttt
gaa gtg gag aag gat ggt gag 1199Tyr Thr Met Thr Leu Leu Asp Leu Phe
Glu Val Glu Lys Asp Gly Glu 385 390 395 aaa gaa gcc ttc aga gag gac
ctt cat aac agg atg ctt cta tgg cat 1247Lys Glu Ala Phe Arg Glu Asp
Leu His Asn Arg Met Leu Leu Trp His 400 405 410 415 ggt tcc agg atg
agt aac tgg gtg gga atc ttg agc cat ggg ctt cga 1295Gly Ser Arg Met
Ser Asn Trp Val Gly Ile Leu Ser His Gly Leu Arg 420 425 430 att gcc
cca cct gaa gct ccc atc aca ggt tac atg ttt ggg aaa gga 1343Ile Ala
Pro Pro Glu Ala Pro Ile Thr Gly Tyr Met Phe Gly Lys Gly 435 440 445
atc tac ttt gct gac atg tct tcc aag agt gcc aat tac tgc ttt gcc
1391Ile Tyr Phe Ala Asp Met Ser Ser Lys Ser Ala Asn Tyr Cys Phe Ala
450 455 460 tct cgc cta aag aat aca gga ctg ctg ctc tta tca gag gta
gct cta 1439Ser Arg Leu Lys Asn Thr Gly Leu Leu Leu Leu Ser Glu Val
Ala Leu 465 470 475 ggt cag tgt aat gaa cta cta gag gcc aat cct aag
gcc gaa gga ttg 1487Gly Gln Cys Asn Glu Leu Leu Glu Ala Asn Pro Lys
Ala Glu Gly Leu 480 485 490 495 ctt caa ggt aaa cat agc acc aag ggg
ctg ggc aag atg gct ccc agt 1535Leu Gln Gly Lys His Ser Thr Lys Gly
Leu Gly Lys Met Ala Pro Ser 500 505 510 tct gcc cac ttc gtc acc ctg
aat ggg agt aca gtg cca tta gga cca 1583Ser Ala His Phe Val Thr Leu
Asn Gly Ser Thr Val Pro Leu Gly Pro 515 520 525 gca agt gac aca gga
att ctg aat cca gat ggt tat acc ctc aac tac 1631Ala Ser Asp Thr Gly
Ile Leu Asn Pro Asp Gly Tyr Thr Leu Asn Tyr 530 535 540 aat gaa tat
att gta tat aac ccc aac cag gtc cgt atg cgg tac ctt 1679Asn Glu Tyr
Ile Val Tyr Asn Pro Asn Gln Val Arg Met Arg Tyr Leu 545 550 555 tta
aag gtt cag ttt aat ttc ctt cag ctg tgg tga atgttgatat 1725Leu Lys
Val Gln Phe Asn Phe Leu Gln Leu Trp 560 565 570 taaataaacc
agagatctga tcttcaagca agaaaataag cagtgttgta cttgtgaatt
1785ttgtgatatt ttatgtaata aaaactgtac aggtctaaaa aaaaaaaaaa aaaaaaaa
18432570PRTHomo sapiens 2Met Ala Ala Arg Arg Arg Arg Ser Thr Gly
Gly Gly Arg Ala Arg Ala 1 5 10 15 Leu Asn Glu Ser Lys Arg Val Asn
Asn Gly Asn Thr Ala Pro Glu Asp 20 25 30 Ser Ser Pro Ala Lys Lys
Thr Arg Arg Cys Gln Arg Gln Glu Ser Lys 35 40 45 Lys Met Pro Val
Ala Gly Gly Lys Ala Asn Lys Asp Arg Thr Glu Asp 50 55 60 Lys Gln
Asp Glu Ser Val Lys Ala Leu Leu Leu Lys Gly Lys Ala Pro 65 70 75 80
Val Asp Pro Glu Cys Thr Ala Lys Val Gly Lys Ala His Val Tyr Cys 85
90 95 Glu Gly Asn Asp Val Tyr Asp Val Met Leu Asn Gln Thr Asn Leu
Gln 100 105 110 Phe Asn Asn Asn Lys Tyr Tyr Leu Ile Gln Leu Leu Glu
Asp Asp Ala 115 120 125 Gln Arg Asn Phe Ser Val Trp Met Arg Trp Gly
Arg Val Gly Lys Met 130 135 140 Gly Gln His Ser Leu Val Ala Cys Ser
Gly Asn Leu Asn Lys Ala Lys 145 150 155 160 Glu Ile Phe Gln Lys Lys
Phe Leu Asp Lys Thr Lys Asn Asn Trp Glu 165 170 175 Asp Arg Glu Lys
Phe Glu Lys Val Pro Gly Lys Tyr Asp Met Leu Gln 180 185 190 Met Asp
Tyr Ala Thr Asn Thr Gln Asp Glu Glu Glu Thr Lys Lys Glu 195 200 205
Glu Ser Leu Lys Ser Pro Leu Lys Pro Glu Ser Gln Leu Asp Leu Arg 210
215 220 Val Gln Glu Leu Ile Lys Leu Ile Cys Asn Val Gln Ala Met Glu
Glu 225 230 235 240 Met Met Met Glu Met Lys Tyr Asn Thr Lys Lys Ala
Pro Leu Gly Lys 245 250 255 Leu Thr Val Ala Gln Ile Lys Ala Gly Tyr
Gln Ser Leu Lys Lys Ile 260 265 270 Glu Asp Cys Ile Arg Ala Gly Gln
His Gly Arg Ala Leu Met Glu Ala 275 280 285 Cys Asn Glu Phe Tyr Thr
Arg Ile Pro His Asp Phe Gly Leu Arg Thr 290 295 300 Pro Pro Leu Ile
Arg Thr Gln Lys Glu Leu Ser Glu Lys Ile Gln Leu 305 310 315 320 Leu
Glu Ala Leu Gly Asp Ile Glu Ile Ala Ile Lys Leu Val Lys Thr 325 330
335 Glu Leu Gln Ser Pro Glu His Pro Leu Asp Gln His Tyr Arg Asn Leu
340 345 350 His Cys Ala Leu Arg Pro Leu Asp His Glu Ser Tyr Glu Phe
Lys Val 355 360 365 Ile Ser Gln Tyr Leu Gln Ser Thr His Ala Pro Thr
His Ser Asp Tyr 370 375 380 Thr Met Thr Leu Leu Asp Leu Phe Glu Val
Glu Lys Asp Gly Glu Lys 385 390 395 400 Glu Ala Phe Arg Glu Asp Leu
His Asn Arg Met Leu Leu Trp His Gly 405 410 415 Ser Arg Met Ser Asn
Trp Val Gly Ile Leu Ser His Gly Leu Arg Ile 420 425 430 Ala Pro Pro
Glu Ala Pro Ile Thr Gly Tyr Met Phe Gly Lys Gly Ile 435 440 445 Tyr
Phe Ala Asp Met Ser Ser Lys Ser Ala Asn Tyr Cys Phe Ala Ser 450 455
460 Arg Leu Lys Asn Thr Gly Leu Leu Leu Leu Ser Glu Val Ala Leu Gly
465 470 475 480 Gln Cys Asn Glu Leu Leu Glu Ala Asn Pro Lys Ala Glu
Gly Leu Leu 485 490 495 Gln Gly Lys His Ser Thr Lys Gly Leu Gly Lys
Met Ala Pro Ser Ser 500 505 510 Ala His Phe Val Thr Leu Asn Gly Ser
Thr Val Pro Leu Gly Pro Ala 515 520 525 Ser Asp Thr Gly Ile Leu Asn
Pro Asp Gly Tyr Thr Leu Asn Tyr Asn 530 535 540 Glu Tyr Ile Val Tyr
Asn Pro Asn Gln Val Arg Met Arg Tyr Leu Leu 545 550 555 560 Lys Val
Gln Phe Asn Phe Leu Gln Leu Trp 565 570 32265DNAHomo
sapiensCDS(242)..(1843)product is Poly ADP Ribose Polymerase; from
uterus tissue 3tgggactggt cgcctgactc ggcctgcccc agcctctgct
tcaccccact ggtggccaaa 60tagccgatgt ctaatccccc acacaagctc atccccggcc
tctgggattg ttgggaattc 120tctccctaat tcacgcctga ggctcatgga
gagttgctag acctgggact gccctgggag 180gcgcacacaa ccaggccggg
tggcagccag gacctctccc atgtccctgc ttttcttggc 240c atg gct cca aag
ccg aag ccc tgg gta cag act gag ggc cct gag aag 289 Met Ala Pro Lys
Pro Lys Pro Trp Val Gln Thr Glu Gly Pro Glu Lys 1 5 10 15 aag aag
ggc cgg cag gca gga agg gag gag gac ccc ttc cgc tcc acc 337Lys Lys
Gly Arg Gln Ala Gly Arg Glu Glu Asp Pro Phe Arg Ser Thr 20 25 30
gct gag gcc ctc aag gcc ata ccc gca gag aag cgc ata atc cgc gtg
385Ala Glu Ala Leu Lys Ala Ile Pro Ala Glu Lys Arg Ile Ile Arg Val
35 40 45 gat cca aca tgt cca ctc agc agc aac ccc ggg acc cag gtg
tat gag 433Asp Pro Thr Cys Pro Leu Ser Ser Asn Pro Gly Thr Gln Val
Tyr Glu 50 55 60 gac tac aac tgc acc ctg aac cag acc aac atc gag
aac aac aac aac 481Asp Tyr Asn Cys Thr Leu Asn Gln Thr Asn Ile Glu
Asn Asn Asn Asn 65 70 75 80 aag ttc tac atc atc cag ctg ctc caa gac
agc aac cgc ttc ttc acc 529Lys Phe Tyr Ile Ile Gln Leu Leu Gln Asp
Ser Asn Arg Phe Phe Thr 85 90 95 tgc tgg aac cgc tgg ggc cgt gtg
gga gag gtc ggc cag tca aag atc 577Cys Trp Asn Arg Trp Gly Arg Val
Gly Glu Val Gly Gln Ser Lys Ile 100 105 110 aac cac ttc aca agg cta
gaa gat gca aag aag gac ttt gag aag aaa 625Asn His Phe Thr Arg Leu
Glu Asp Ala Lys Lys Asp Phe Glu Lys Lys 115 120 125 ttt cgg gaa aag
acc aag aac aac tgg gca gag cgg gac cac ttt gtg 673Phe Arg Glu Lys
Thr Lys Asn Asn Trp Ala Glu Arg Asp His Phe Val 130 135 140 tct cac
ccg ggc aag tac aca ctt atc gaa gta cag gca gag gat gag 721Ser His
Pro Gly Lys Tyr Thr Leu Ile Glu Val Gln Ala Glu Asp Glu 145 150 155
160 gcc cag gaa gct gtg gtg aag gtg gac aga ggc cca gtg agg act gtg
769Ala Gln Glu Ala Val Val Lys Val Asp Arg Gly Pro Val Arg Thr Val
165 170 175 act aag cgg gtg cag ccc tgc tcc ctg gac cca gcc acg cag
aag ctc 817Thr Lys Arg Val Gln Pro Cys Ser Leu Asp Pro Ala Thr Gln
Lys Leu 180 185 190 atc act aac atc ttc agc aag gag atg ttc aag aac
acc atg gcc ctc 865Ile Thr Asn Ile Phe Ser Lys Glu Met Phe Lys Asn
Thr Met Ala Leu 195 200 205 atg gac ctg gat gtg aag aag atg ccc ctg
gga aag ctg agc aag caa 913Met Asp Leu Asp Val Lys Lys Met Pro Leu
Gly Lys Leu Ser Lys Gln 210 215 220 cag att gca cgg ggt ttc gag gcc
ttg gag gcg ctg gag gag gcc ctg 961Gln Ile Ala Arg Gly Phe Glu Ala
Leu Glu Ala Leu Glu Glu Ala Leu 225 230 235 240 aaa ggc ccc acg gat
ggt ggc caa agc ctg gag gag ctg tcc tca cac 1009Lys Gly Pro Thr Asp
Gly Gly Gln Ser Leu Glu Glu Leu Ser Ser His 245 250 255 ttt tac acc
gtc atc ccg cac aac ttc ggc cac agc cag ccc ccg ccc 1057Phe Tyr Thr
Val Ile Pro His Asn Phe Gly His Ser Gln Pro Pro Pro 260 265 270 atc
aat tcc cct gag ctt ctg cag gcc aag aag gac atg ctg ctg gtg 1105Ile
Asn Ser Pro Glu Leu Leu Gln Ala Lys Lys Asp Met Leu Leu Val 275 280
285 ctg gcg gac atc gag ctg gcc cag gcc ctg cag gca gtc tct gag cag
1153Leu Ala Asp Ile Glu Leu Ala Gln Ala Leu Gln Ala Val Ser Glu Gln
290 295 300 gag aag acg gtg gag gag gtg cca cac ccc ctg gac cga gac
tac cag 1201Glu Lys Thr Val Glu Glu Val Pro His Pro Leu Asp Arg Asp
Tyr Gln 305 310 315 320 ctt ctc aag tgc cag ctg cag ctg cta gac tct
gga gca cct gag tac 1249Leu Leu Lys Cys Gln Leu Gln Leu Leu Asp Ser
Gly Ala Pro Glu Tyr 325 330 335 aag gtg ata cag acc tac tta gaa cag
act ggc agc aac cac agg tgc 1297Lys Val Ile Gln Thr Tyr Leu Glu Gln
Thr Gly Ser Asn His Arg Cys 340 345 350 cct aca ctt caa cac atc tgg
aaa gta aac caa gaa ggg gag gaa gac 1345Pro Thr Leu Gln His Ile Trp
Lys Val Asn Gln Glu Gly Glu Glu Asp 355 360 365 aga ttc cag gcc cac
tcc aaa ctg ggt aat cgg aag ctg ctg tgg cat 1393Arg Phe Gln Ala His
Ser Lys Leu Gly Asn Arg Lys Leu Leu Trp His 370 375 380 ggc acc aac
atg gcc gtg gtg gcc gcc atc ctc act agt ggg ctc cgc
1441Gly Thr Asn Met Ala Val Val Ala Ala Ile Leu Thr Ser Gly Leu Arg
385 390 395 400 atc atg cca cat tct ggt ggg cgt gtt ggc aag ggc atc
tac ttt gcc 1489Ile Met Pro His Ser Gly Gly Arg Val Gly Lys Gly Ile
Tyr Phe Ala 405 410 415 tca gag aac agc aag tca gct gga tat gtt att
ggc atg aag tgt ggg 1537Ser Glu Asn Ser Lys Ser Ala Gly Tyr Val Ile
Gly Met Lys Cys Gly 420 425 430 gcc cac cat gtc ggc tac atg ttc ctg
ggt gag gtg gcc ctg ggc aga 1585Ala His His Val Gly Tyr Met Phe Leu
Gly Glu Val Ala Leu Gly Arg 435 440 445 gag cac cat atc aac acg gac
aac ccc agc ttg aag agc cca cct cct 1633Glu His His Ile Asn Thr Asp
Asn Pro Ser Leu Lys Ser Pro Pro Pro 450 455 460 ggc ttc gac agt gtc
att gcc cga ggc cac acc gag cct gat ccg acc 1681Gly Phe Asp Ser Val
Ile Ala Arg Gly His Thr Glu Pro Asp Pro Thr 465 470 475 480 cag gac
act gag ttg gag ctg gat ggc cag caa gtg gtg gtg ccc cag 1729Gln Asp
Thr Glu Leu Glu Leu Asp Gly Gln Gln Val Val Val Pro Gln 485 490 495
ggc cag cct gtg ccc tgc cca gag ttc agc agc tcc aca ttc tcc cag
1777Gly Gln Pro Val Pro Cys Pro Glu Phe Ser Ser Ser Thr Phe Ser Gln
500 505 510 agc gag tac ctc atc tac cag gag agc cag tgt cgc ctg cgc
tac ctg 1825Ser Glu Tyr Leu Ile Tyr Gln Glu Ser Gln Cys Arg Leu Arg
Tyr Leu 515 520 525 ctg gag gtc cac ctc tga gtgcccgccc tgtcccccgg
ggtcctgcaa 1873Leu Glu Val His Leu 530 ggctggactg tgatcttcaa
tcatcctgcc catctctggt acccctatat cactcctttt 1933tttcaagaat
acaatacgtt gttgttaact atagtcacca tgctgtacaa gatccctgaa
1993cttatgcctc ctaactgaaa ttttgtattc tttgacacat ctgcccagtc
cctctcctcc 2053cagcccatgg taaccagcat ttgactcttt acttgtataa
gggcagcttt tataggttcc 2113acatgtaagt gagatcatgc agtgtttgtc
tttctgtgcc tggcttattt cactcagcat 2173aatgtgcacc gggttcaccc
atgttttcat aaatgacaag atttcctcct ttaaaaaaaa 2233aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aa 22654533PRTHomo sapiens 4Met Ala Pro Lys
Pro Lys Pro Trp Val Gln Thr Glu Gly Pro Glu Lys 1 5 10 15 Lys Lys
Gly Arg Gln Ala Gly Arg Glu Glu Asp Pro Phe Arg Ser Thr 20 25 30
Ala Glu Ala Leu Lys Ala Ile Pro Ala Glu Lys Arg Ile Ile Arg Val 35
40 45 Asp Pro Thr Cys Pro Leu Ser Ser Asn Pro Gly Thr Gln Val Tyr
Glu 50 55 60 Asp Tyr Asn Cys Thr Leu Asn Gln Thr Asn Ile Glu Asn
Asn Asn Asn 65 70 75 80 Lys Phe Tyr Ile Ile Gln Leu Leu Gln Asp Ser
Asn Arg Phe Phe Thr 85 90 95 Cys Trp Asn Arg Trp Gly Arg Val Gly
Glu Val Gly Gln Ser Lys Ile 100 105 110 Asn His Phe Thr Arg Leu Glu
Asp Ala Lys Lys Asp Phe Glu Lys Lys 115 120 125 Phe Arg Glu Lys Thr
Lys Asn Asn Trp Ala Glu Arg Asp His Phe Val 130 135 140 Ser His Pro
Gly Lys Tyr Thr Leu Ile Glu Val Gln Ala Glu Asp Glu 145 150 155 160
Ala Gln Glu Ala Val Val Lys Val Asp Arg Gly Pro Val Arg Thr Val 165
170 175 Thr Lys Arg Val Gln Pro Cys Ser Leu Asp Pro Ala Thr Gln Lys
Leu 180 185 190 Ile Thr Asn Ile Phe Ser Lys Glu Met Phe Lys Asn Thr
Met Ala Leu 195 200 205 Met Asp Leu Asp Val Lys Lys Met Pro Leu Gly
Lys Leu Ser Lys Gln 210 215 220 Gln Ile Ala Arg Gly Phe Glu Ala Leu
Glu Ala Leu Glu Glu Ala Leu 225 230 235 240 Lys Gly Pro Thr Asp Gly
Gly Gln Ser Leu Glu Glu Leu Ser Ser His 245 250 255 Phe Tyr Thr Val
Ile Pro His Asn Phe Gly His Ser Gln Pro Pro Pro 260 265 270 Ile Asn
Ser Pro Glu Leu Leu Gln Ala Lys Lys Asp Met Leu Leu Val 275 280 285
Leu Ala Asp Ile Glu Leu Ala Gln Ala Leu Gln Ala Val Ser Glu Gln 290
295 300 Glu Lys Thr Val Glu Glu Val Pro His Pro Leu Asp Arg Asp Tyr
Gln 305 310 315 320 Leu Leu Lys Cys Gln Leu Gln Leu Leu Asp Ser Gly
Ala Pro Glu Tyr 325 330 335 Lys Val Ile Gln Thr Tyr Leu Glu Gln Thr
Gly Ser Asn His Arg Cys 340 345 350 Pro Thr Leu Gln His Ile Trp Lys
Val Asn Gln Glu Gly Glu Glu Asp 355 360 365 Arg Phe Gln Ala His Ser
Lys Leu Gly Asn Arg Lys Leu Leu Trp His 370 375 380 Gly Thr Asn Met
Ala Val Val Ala Ala Ile Leu Thr Ser Gly Leu Arg 385 390 395 400 Ile
Met Pro His Ser Gly Gly Arg Val Gly Lys Gly Ile Tyr Phe Ala 405 410
415 Ser Glu Asn Ser Lys Ser Ala Gly Tyr Val Ile Gly Met Lys Cys Gly
420 425 430 Ala His His Val Gly Tyr Met Phe Leu Gly Glu Val Ala Leu
Gly Arg 435 440 445 Glu His His Ile Asn Thr Asp Asn Pro Ser Leu Lys
Ser Pro Pro Pro 450 455 460 Gly Phe Asp Ser Val Ile Ala Arg Gly His
Thr Glu Pro Asp Pro Thr 465 470 475 480 Gln Asp Thr Glu Leu Glu Leu
Asp Gly Gln Gln Val Val Val Pro Gln 485 490 495 Gly Gln Pro Val Pro
Cys Pro Glu Phe Ser Ser Ser Thr Phe Ser Gln 500 505 510 Ser Glu Tyr
Leu Ile Tyr Gln Glu Ser Gln Cys Arg Leu Arg Tyr Leu 515 520 525 Leu
Glu Val His Leu 530 52265DNAHomo sapiensCDS(221)..(1843)product is
Poly ADP Ribose Polymerase; from uterus tissue 5tgggactggt
cgcctgactc ggcctgcccc agcctctgct tcaccccact ggtggccaaa 60tagccgatgt
ctaatccccc acacaagctc atccccggcc tctgggattg ttgggaattc
120tctccctaat tcacgcctga ggctcatgga gagttgctag acctgggact
gccctgggag 180gcgcacacaa ccaggccggg tggcagccag gacctctccc atg tcc
ctg ctt ttc 235 Met Ser Leu Leu Phe 1 5 ttg gcc atg gct cca aag ccg
aag ccc tgg gta cag act gag ggc cct 283Leu Ala Met Ala Pro Lys Pro
Lys Pro Trp Val Gln Thr Glu Gly Pro 10 15 20 gag aag aag aag ggc
cgg cag gca gga agg gag gag gac ccc ttc cgc 331Glu Lys Lys Lys Gly
Arg Gln Ala Gly Arg Glu Glu Asp Pro Phe Arg 25 30 35 tcc acc gct
gag gcc ctc aag gcc ata ccc gca gag aag cgc ata atc 379Ser Thr Ala
Glu Ala Leu Lys Ala Ile Pro Ala Glu Lys Arg Ile Ile 40 45 50 cgc
gtg gat cca aca tgt cca ctc agc agc aac ccc ggg acc cag gtg 427Arg
Val Asp Pro Thr Cys Pro Leu Ser Ser Asn Pro Gly Thr Gln Val 55 60
65 tat gag gac tac aac tgc acc ctg aac cag acc aac atc gag aac aac
475Tyr Glu Asp Tyr Asn Cys Thr Leu Asn Gln Thr Asn Ile Glu Asn Asn
70 75 80 85 aac aac aag ttc tac atc atc cag ctg ctc caa gac agc aac
cgc ttc 523Asn Asn Lys Phe Tyr Ile Ile Gln Leu Leu Gln Asp Ser Asn
Arg Phe 90 95 100 ttc acc tgc tgg aac cgc tgg ggc cgt gtg gga gag
gtc ggc cag tca 571Phe Thr Cys Trp Asn Arg Trp Gly Arg Val Gly Glu
Val Gly Gln Ser 105 110 115 aag atc aac cac ttc aca agg cta gaa gat
gca aag aag gac ttt gag 619Lys Ile Asn His Phe Thr Arg Leu Glu Asp
Ala Lys Lys Asp Phe Glu 120 125 130 aag aaa ttt cgg gaa aag acc aag
aac aac tgg gca gag cgg gac cac 667Lys Lys Phe Arg Glu Lys Thr Lys
Asn Asn Trp Ala Glu Arg Asp His 135 140 145 ttt gtg tct cac ccg ggc
aag tac aca ctt atc gaa gta cag gca gag 715Phe Val Ser His Pro Gly
Lys Tyr Thr Leu Ile Glu Val Gln Ala Glu 150 155 160 165 gat gag gcc
cag gaa gct gtg gtg aag gtg gac aga ggc cca gtg agg 763Asp Glu Ala
Gln Glu Ala Val Val Lys Val Asp Arg Gly Pro Val Arg 170 175 180 act
gtg act aag cgg gtg cag ccc tgc tcc ctg gac cca gcc acg cag 811Thr
Val Thr Lys Arg Val Gln Pro Cys Ser Leu Asp Pro Ala Thr Gln 185 190
195 aag ctc atc act aac atc ttc agc aag gag atg ttc aag aac acc atg
859Lys Leu Ile Thr Asn Ile Phe Ser Lys Glu Met Phe Lys Asn Thr Met
200 205 210 gcc ctc atg gac ctg gat gtg aag aag atg ccc ctg gga aag
ctg agc 907Ala Leu Met Asp Leu Asp Val Lys Lys Met Pro Leu Gly Lys
Leu Ser 215 220 225 aag caa cag att gca cgg ggt ttc gag gcc ttg gag
gcg ctg gag gag 955Lys Gln Gln Ile Ala Arg Gly Phe Glu Ala Leu Glu
Ala Leu Glu Glu 230 235 240 245 gcc ctg aaa ggc ccc acg gat ggt ggc
caa agc ctg gag gag ctg tcc 1003Ala Leu Lys Gly Pro Thr Asp Gly Gly
Gln Ser Leu Glu Glu Leu Ser 250 255 260 tca cac ttt tac acc gtc atc
ccg cac aac ttc ggc cac agc cag ccc 1051Ser His Phe Tyr Thr Val Ile
Pro His Asn Phe Gly His Ser Gln Pro 265 270 275 ccg ccc atc aat tcc
cct gag ctt ctg cag gcc aag aag gac atg ctg 1099Pro Pro Ile Asn Ser
Pro Glu Leu Leu Gln Ala Lys Lys Asp Met Leu 280 285 290 ctg gtg ctg
gcg gac atc gag ctg gcc cag gcc ctg cag gca gtc tct 1147Leu Val Leu
Ala Asp Ile Glu Leu Ala Gln Ala Leu Gln Ala Val Ser 295 300 305 gag
cag gag aag acg gtg gag gag gtg cca cac ccc ctg gac cga gac 1195Glu
Gln Glu Lys Thr Val Glu Glu Val Pro His Pro Leu Asp Arg Asp 310 315
320 325 tac cag ctt ctc aag tgc cag ctg cag ctg cta gac tct gga gca
cct 1243Tyr Gln Leu Leu Lys Cys Gln Leu Gln Leu Leu Asp Ser Gly Ala
Pro 330 335 340 gag tac aag gtg ata cag acc tac tta gaa cag act ggc
agc aac cac 1291Glu Tyr Lys Val Ile Gln Thr Tyr Leu Glu Gln Thr Gly
Ser Asn His 345 350 355 agg tgc cct aca ctt caa cac atc tgg aaa gta
aac caa gaa ggg gag 1339Arg Cys Pro Thr Leu Gln His Ile Trp Lys Val
Asn Gln Glu Gly Glu 360 365 370 gaa gac aga ttc cag gcc cac tcc aaa
ctg ggt aat cgg aag ctg ctg 1387Glu Asp Arg Phe Gln Ala His Ser Lys
Leu Gly Asn Arg Lys Leu Leu 375 380 385 tgg cat ggc acc aac atg gcc
gtg gtg gcc gcc atc ctc act agt ggg 1435Trp His Gly Thr Asn Met Ala
Val Val Ala Ala Ile Leu Thr Ser Gly 390 395 400 405 ctc cgc atc atg
cca cat tct ggt ggg cgt gtt ggc aag ggc atc tac 1483Leu Arg Ile Met
Pro His Ser Gly Gly Arg Val Gly Lys Gly Ile Tyr 410 415 420 ttt gcc
tca gag aac agc aag tca gct gga tat gtt att ggc atg aag 1531Phe Ala
Ser Glu Asn Ser Lys Ser Ala Gly Tyr Val Ile Gly Met Lys 425 430 435
tgt ggg gcc cac cat gtc ggc tac atg ttc ctg ggt gag gtg gcc ctg
1579Cys Gly Ala His His Val Gly Tyr Met Phe Leu Gly Glu Val Ala Leu
440 445 450 ggc aga gag cac cat atc aac acg gac aac ccc agc ttg aag
agc cca 1627Gly Arg Glu His His Ile Asn Thr Asp Asn Pro Ser Leu Lys
Ser Pro 455 460 465 cct cct ggc ttc gac agt gtc att gcc cga ggc cac
acc gag cct gat 1675Pro Pro Gly Phe Asp Ser Val Ile Ala Arg Gly His
Thr Glu Pro Asp 470 475 480 485 ccg acc cag gac act gag ttg gag ctg
gat ggc cag caa gtg gtg gtg 1723Pro Thr Gln Asp Thr Glu Leu Glu Leu
Asp Gly Gln Gln Val Val Val 490 495 500 ccc cag ggc cag cct gtg ccc
tgc cca gag ttc agc agc tcc aca ttc 1771Pro Gln Gly Gln Pro Val Pro
Cys Pro Glu Phe Ser Ser Ser Thr Phe 505 510 515 tcc cag agc gag tac
ctc atc tac cag gag agc cag tgt cgc ctg cgc 1819Ser Gln Ser Glu Tyr
Leu Ile Tyr Gln Glu Ser Gln Cys Arg Leu Arg 520 525 530 tac ctg ctg
gag gtc cac ctc tga gtgcccgccc tgtcccccgg ggtcctgcaa 1873Tyr Leu
Leu Glu Val His Leu 535 540 ggctggactg tgatcttcaa tcatcctgcc
catctctggt acccctatat cactcctttt 1933tttcaagaat acaatacgtt
gttgttaact atagtcacca tgctgtacaa gatccctgaa 1993cttatgcctc
ctaactgaaa ttttgtattc tttgacacat ctgcccagtc cctctcctcc
2053cagcccatgg taaccagcat ttgactcttt acttgtataa gggcagcttt
tataggttcc 2113acatgtaagt gagatcatgc agtgtttgtc tttctgtgcc
tggcttattt cactcagcat 2173aatgtgcacc gggttcaccc atgttttcat
aaatgacaag atttcctcct ttaaaaaaaa 2233aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aa 22656540PRTHomo sapiens 6Met Ser Leu Leu Phe Leu Ala
Met Ala Pro Lys Pro Lys Pro Trp Val 1 5 10 15 Gln Thr Glu Gly Pro
Glu Lys Lys Lys Gly Arg Gln Ala Gly Arg Glu 20 25 30 Glu Asp Pro
Phe Arg Ser Thr Ala Glu Ala Leu Lys Ala Ile Pro Ala 35 40 45 Glu
Lys Arg Ile Ile Arg Val Asp Pro Thr Cys Pro Leu Ser Ser Asn 50 55
60 Pro Gly Thr Gln Val Tyr Glu Asp Tyr Asn Cys Thr Leu Asn Gln Thr
65 70 75 80 Asn Ile Glu Asn Asn Asn Asn Lys Phe Tyr Ile Ile Gln Leu
Leu Gln 85 90 95 Asp Ser Asn Arg Phe Phe Thr Cys Trp Asn Arg Trp
Gly Arg Val Gly 100 105 110 Glu Val Gly Gln Ser Lys Ile Asn His Phe
Thr Arg Leu Glu Asp Ala 115 120 125 Lys Lys Asp Phe Glu Lys Lys Phe
Arg Glu Lys Thr Lys Asn Asn Trp 130 135 140 Ala Glu Arg Asp His Phe
Val Ser His Pro Gly Lys Tyr Thr Leu Ile 145 150 155 160 Glu Val Gln
Ala Glu Asp Glu Ala Gln Glu Ala Val Val Lys Val Asp 165 170 175 Arg
Gly Pro Val Arg Thr Val Thr Lys Arg Val Gln Pro Cys Ser Leu 180 185
190 Asp Pro Ala Thr Gln Lys Leu Ile Thr Asn Ile Phe Ser Lys Glu Met
195 200 205 Phe Lys Asn Thr Met Ala Leu Met Asp Leu Asp Val Lys Lys
Met Pro 210 215 220 Leu Gly Lys Leu Ser Lys Gln Gln Ile Ala Arg Gly
Phe Glu Ala Leu 225 230 235 240 Glu Ala Leu Glu Glu Ala Leu Lys Gly
Pro Thr Asp Gly Gly Gln Ser 245 250 255 Leu Glu Glu Leu Ser Ser His
Phe Tyr Thr Val Ile Pro His Asn Phe 260 265 270 Gly His Ser Gln Pro
Pro Pro Ile Asn Ser Pro Glu Leu Leu Gln Ala 275 280 285 Lys Lys Asp
Met Leu Leu Val Leu Ala Asp Ile Glu Leu Ala Gln Ala 290 295 300 Leu
Gln Ala Val Ser Glu Gln Glu Lys Thr Val Glu Glu Val Pro His 305 310
315 320 Pro Leu Asp Arg Asp Tyr Gln Leu Leu Lys Cys Gln Leu Gln Leu
Leu 325 330 335 Asp Ser Gly Ala Pro Glu Tyr Lys Val Ile Gln Thr Tyr
Leu Glu Gln 340 345 350 Thr Gly Ser Asn His Arg Cys Pro Thr Leu Gln
His Ile Trp Lys Val 355 360 365 Asn Gln Glu Gly Glu Glu Asp Arg Phe
Gln Ala His Ser
Lys Leu Gly 370 375 380 Asn Arg Lys Leu Leu Trp His Gly Thr Asn Met
Ala Val Val Ala Ala 385 390 395 400 Ile Leu Thr Ser Gly Leu Arg Ile
Met Pro His Ser Gly Gly Arg Val 405 410 415 Gly Lys Gly Ile Tyr Phe
Ala Ser Glu Asn Ser Lys Ser Ala Gly Tyr 420 425 430 Val Ile Gly Met
Lys Cys Gly Ala His His Val Gly Tyr Met Phe Leu 435 440 445 Gly Glu
Val Ala Leu Gly Arg Glu His His Ile Asn Thr Asp Asn Pro 450 455 460
Ser Leu Lys Ser Pro Pro Pro Gly Phe Asp Ser Val Ile Ala Arg Gly 465
470 475 480 His Thr Glu Pro Asp Pro Thr Gln Asp Thr Glu Leu Glu Leu
Asp Gly 485 490 495 Gln Gln Val Val Val Pro Gln Gly Gln Pro Val Pro
Cys Pro Glu Phe 500 505 510 Ser Ser Ser Thr Phe Ser Gln Ser Glu Tyr
Leu Ile Tyr Gln Glu Ser 515 520 525 Gln Cys Arg Leu Arg Tyr Leu Leu
Glu Val His Leu 530 535 540 71740DNAMus musculusCDS(112)..(1710)
7cccggctttc actttttctg ctgcctcggg gaacacctcg agccaactgc ttcctaactc
60agggtgggca gaactgacgg gatctaagct tctgcatctc tgaggagaac c atg gct
117 Met Ala 1 cca aaa cga aag gcc tct gtg cag act gag ggc tcc aag
aag cag cga 165Pro Lys Arg Lys Ala Ser Val Gln Thr Glu Gly Ser Lys
Lys Gln Arg 5 10 15 caa ggg aca gag gag gag gac agc ttc cgg tcc act
gcc gag gct ctc 213Gln Gly Thr Glu Glu Glu Asp Ser Phe Arg Ser Thr
Ala Glu Ala Leu 20 25 30 aga gca gca cct gct gat aat cgg gtc atc
cgt gtg gac ccc tca tgt 261Arg Ala Ala Pro Ala Asp Asn Arg Val Ile
Arg Val Asp Pro Ser Cys 35 40 45 50 cca ttc agc cgg aac ccc ggg ata
cag gtc cac gag gac tat gac tgt 309Pro Phe Ser Arg Asn Pro Gly Ile
Gln Val His Glu Asp Tyr Asp Cys 55 60 65 acc ctg aac cag acc aac
atc ggc aac aac aac aac aag ttc tat att 357Thr Leu Asn Gln Thr Asn
Ile Gly Asn Asn Asn Asn Lys Phe Tyr Ile 70 75 80 atc caa ctg ctg
gag gag ggt agt cgc ttc ttc tgc tgg aat cgc tgg 405Ile Gln Leu Leu
Glu Glu Gly Ser Arg Phe Phe Cys Trp Asn Arg Trp 85 90 95 ggc cgc
gtg gga gag gtg ggc cag agc aag atg aac cac ttc acc tgc 453Gly Arg
Val Gly Glu Val Gly Gln Ser Lys Met Asn His Phe Thr Cys 100 105 110
ctg gaa gat gca aag aag gac ttt aag aag aaa ttt tgg gag aag act
501Leu Glu Asp Ala Lys Lys Asp Phe Lys Lys Lys Phe Trp Glu Lys Thr
115 120 125 130 aaa aac aaa tgg gag gag cgg gac cgt ttt gtg gcc cag
ccc aac aag 549Lys Asn Lys Trp Glu Glu Arg Asp Arg Phe Val Ala Gln
Pro Asn Lys 135 140 145 tac aca ctt ata gaa gtc cag gga gaa gca gag
agc caa gag gct gta 597Tyr Thr Leu Ile Glu Val Gln Gly Glu Ala Glu
Ser Gln Glu Ala Val 150 155 160 gtg aag gcc tta tct ccc cag gtg gac
agc ggc cct gtg agg acc gtg 645Val Lys Ala Leu Ser Pro Gln Val Asp
Ser Gly Pro Val Arg Thr Val 165 170 175 gtc aag ccc tgc tcc cta gac
cct gcc acc cag aac ctt atc acc aac 693Val Lys Pro Cys Ser Leu Asp
Pro Ala Thr Gln Asn Leu Ile Thr Asn 180 185 190 atc ttc agc aaa gag
atg ttc aag aac gca atg acc ctc atg aac ctg 741Ile Phe Ser Lys Glu
Met Phe Lys Asn Ala Met Thr Leu Met Asn Leu 195 200 205 210 gat gtg
aag aag atg ccc ttg gga aag ctg acc aag cag cag att gcc 789Asp Val
Lys Lys Met Pro Leu Gly Lys Leu Thr Lys Gln Gln Ile Ala 215 220 225
cgt ggc ttc gag gcc ttg gaa gct cta gag gag gcc atg aaa aac ccc
837Arg Gly Phe Glu Ala Leu Glu Ala Leu Glu Glu Ala Met Lys Asn Pro
230 235 240 aca ggg gat ggc cag agc ctg gaa gag ctc tcc tcc tgc ttc
tac act 885Thr Gly Asp Gly Gln Ser Leu Glu Glu Leu Ser Ser Cys Phe
Tyr Thr 245 250 255 gtc atc cca cac aac ttc ggc cgc agc cga ccc ccg
ccc atc aac tcc 933Val Ile Pro His Asn Phe Gly Arg Ser Arg Pro Pro
Pro Ile Asn Ser 260 265 270 cct gat gtg ctt cag gcc aag aag gac atg
ctg ctg gtg cta gcg gac 981Pro Asp Val Leu Gln Ala Lys Lys Asp Met
Leu Leu Val Leu Ala Asp 275 280 285 290 atc gag ttg gcg cag acc ttg
cag gca gcc cct ggg gag gag gag gag 1029Ile Glu Leu Ala Gln Thr Leu
Gln Ala Ala Pro Gly Glu Glu Glu Glu 295 300 305 aaa gtg gaa gag gtg
cca cac cca ctg gat cga gac tac cag ctc ctc 1077Lys Val Glu Glu Val
Pro His Pro Leu Asp Arg Asp Tyr Gln Leu Leu 310 315 320 agg tgc cag
ctt caa ctg ctg gac tcc ggg gag tcc gag tac aag gca 1125Arg Cys Gln
Leu Gln Leu Leu Asp Ser Gly Glu Ser Glu Tyr Lys Ala 325 330 335 ata
cag acc tac ctg aaa cag act ggc aac agc tac agg tgc cca aac 1173Ile
Gln Thr Tyr Leu Lys Gln Thr Gly Asn Ser Tyr Arg Cys Pro Asn 340 345
350 ctg cgg cat gtt tgg aaa gtg aac cga gaa ggg gag gga gac agg ttc
1221Leu Arg His Val Trp Lys Val Asn Arg Glu Gly Glu Gly Asp Arg Phe
355 360 365 370 cag gcc cac tcc aaa ctg ggc aat cgg agg ctg ctg tgg
cac ggc acc 1269Gln Ala His Ser Lys Leu Gly Asn Arg Arg Leu Leu Trp
His Gly Thr 375 380 385 aat gtg gcc gtg gtg gct gcc atc ctc acc agt
ggg ctc cga atc atg 1317Asn Val Ala Val Val Ala Ala Ile Leu Thr Ser
Gly Leu Arg Ile Met 390 395 400 cca cac tcg ggt ggt cgt gtt ggc aag
ggt att tat ttt gcc tct gag 1365Pro His Ser Gly Gly Arg Val Gly Lys
Gly Ile Tyr Phe Ala Ser Glu 405 410 415 aac agc aag tca gct ggc tat
gtt acc acc atg cac tgt ggg ggc cac 1413Asn Ser Lys Ser Ala Gly Tyr
Val Thr Thr Met His Cys Gly Gly His 420 425 430 cag gtg ggc tac atg
ttc ctg ggc gag gtg gcc ctc ggc aaa gag cac 1461Gln Val Gly Tyr Met
Phe Leu Gly Glu Val Ala Leu Gly Lys Glu His 435 440 445 450 cac atc
acc atc gat gac ccc agc ttg aag agt cca ccc cct ggc ttt 1509His Ile
Thr Ile Asp Asp Pro Ser Leu Lys Ser Pro Pro Pro Gly Phe 455 460 465
gac agc gtc atc gcc cga ggc caa acc gag ccg gat ccc gcc cag gac
1557Asp Ser Val Ile Ala Arg Gly Gln Thr Glu Pro Asp Pro Ala Gln Asp
470 475 480 att gaa ctt gaa ctg gat ggg cag ccg gtg gtg gtg ccc caa
ggc ccg 1605Ile Glu Leu Glu Leu Asp Gly Gln Pro Val Val Val Pro Gln
Gly Pro 485 490 495 cct gtg cag tgc ccg tca ttc aaa agc tcc agc ttc
agc cag agt gaa 1653Pro Val Gln Cys Pro Ser Phe Lys Ser Ser Ser Phe
Ser Gln Ser Glu 500 505 510 tac ctc ata tac aag gag agc cag tgt cgc
ctg cgc tac ctg ctg gag 1701Tyr Leu Ile Tyr Lys Glu Ser Gln Cys Arg
Leu Arg Tyr Leu Leu Glu 515 520 525 530 att cac ctc taagctgctt
gccctcccta ggtccaagcc 1740Ile His Leu 8533PRTMus musculus 8Met Ala
Pro Lys Arg Lys Ala Ser Val Gln Thr Glu Gly Ser Lys Lys 1 5 10 15
Gln Arg Gln Gly Thr Glu Glu Glu Asp Ser Phe Arg Ser Thr Ala Glu 20
25 30 Ala Leu Arg Ala Ala Pro Ala Asp Asn Arg Val Ile Arg Val Asp
Pro 35 40 45 Ser Cys Pro Phe Ser Arg Asn Pro Gly Ile Gln Val His
Glu Asp Tyr 50 55 60 Asp Cys Thr Leu Asn Gln Thr Asn Ile Gly Asn
Asn Asn Asn Lys Phe 65 70 75 80 Tyr Ile Ile Gln Leu Leu Glu Glu Gly
Ser Arg Phe Phe Cys Trp Asn 85 90 95 Arg Trp Gly Arg Val Gly Glu
Val Gly Gln Ser Lys Met Asn His Phe 100 105 110 Thr Cys Leu Glu Asp
Ala Lys Lys Asp Phe Lys Lys Lys Phe Trp Glu 115 120 125 Lys Thr Lys
Asn Lys Trp Glu Glu Arg Asp Arg Phe Val Ala Gln Pro 130 135 140 Asn
Lys Tyr Thr Leu Ile Glu Val Gln Gly Glu Ala Glu Ser Gln Glu 145 150
155 160 Ala Val Val Lys Ala Leu Ser Pro Gln Val Asp Ser Gly Pro Val
Arg 165 170 175 Thr Val Val Lys Pro Cys Ser Leu Asp Pro Ala Thr Gln
Asn Leu Ile 180 185 190 Thr Asn Ile Phe Ser Lys Glu Met Phe Lys Asn
Ala Met Thr Leu Met 195 200 205 Asn Leu Asp Val Lys Lys Met Pro Leu
Gly Lys Leu Thr Lys Gln Gln 210 215 220 Ile Ala Arg Gly Phe Glu Ala
Leu Glu Ala Leu Glu Glu Ala Met Lys 225 230 235 240 Asn Pro Thr Gly
Asp Gly Gln Ser Leu Glu Glu Leu Ser Ser Cys Phe 245 250 255 Tyr Thr
Val Ile Pro His Asn Phe Gly Arg Ser Arg Pro Pro Pro Ile 260 265 270
Asn Ser Pro Asp Val Leu Gln Ala Lys Lys Asp Met Leu Leu Val Leu 275
280 285 Ala Asp Ile Glu Leu Ala Gln Thr Leu Gln Ala Ala Pro Gly Glu
Glu 290 295 300 Glu Glu Lys Val Glu Glu Val Pro His Pro Leu Asp Arg
Asp Tyr Gln 305 310 315 320 Leu Leu Arg Cys Gln Leu Gln Leu Leu Asp
Ser Gly Glu Ser Glu Tyr 325 330 335 Lys Ala Ile Gln Thr Tyr Leu Lys
Gln Thr Gly Asn Ser Tyr Arg Cys 340 345 350 Pro Asn Leu Arg His Val
Trp Lys Val Asn Arg Glu Gly Glu Gly Asp 355 360 365 Arg Phe Gln Ala
His Ser Lys Leu Gly Asn Arg Arg Leu Leu Trp His 370 375 380 Gly Thr
Asn Val Ala Val Val Ala Ala Ile Leu Thr Ser Gly Leu Arg 385 390 395
400 Ile Met Pro His Ser Gly Gly Arg Val Gly Lys Gly Ile Tyr Phe Ala
405 410 415 Ser Glu Asn Ser Lys Ser Ala Gly Tyr Val Thr Thr Met His
Cys Gly 420 425 430 Gly His Gln Val Gly Tyr Met Phe Leu Gly Glu Val
Ala Leu Gly Lys 435 440 445 Glu His His Ile Thr Ile Asp Asp Pro Ser
Leu Lys Ser Pro Pro Pro 450 455 460 Gly Phe Asp Ser Val Ile Ala Arg
Gly Gln Thr Glu Pro Asp Pro Ala 465 470 475 480 Gln Asp Ile Glu Leu
Glu Leu Asp Gly Gln Pro Val Val Val Pro Gln 485 490 495 Gly Pro Pro
Val Gln Cys Pro Ser Phe Lys Ser Ser Ser Phe Ser Gln 500 505 510 Ser
Glu Tyr Leu Ile Tyr Lys Glu Ser Gln Cys Arg Leu Arg Tyr Leu 515 520
525 Leu Glu Ile His Leu 530 91587DNAMus musculusCDS(1)..(1584) 9atg
gct cca aaa cga aag gcc tct gtg cag act gag ggc tcc aag aag 48Met
Ala Pro Lys Arg Lys Ala Ser Val Gln Thr Glu Gly Ser Lys Lys 1 5 10
15 cag cga caa ggg aca gag gag gag gac agc ttc cgg tcc act gcc gag
96Gln Arg Gln Gly Thr Glu Glu Glu Asp Ser Phe Arg Ser Thr Ala Glu
20 25 30 gct ctc aga gca gca cct gct gat aat cgg gtc atc cgt gtg
gac ccc 144Ala Leu Arg Ala Ala Pro Ala Asp Asn Arg Val Ile Arg Val
Asp Pro 35 40 45 tca tgt cca ttc agc cgg aac ccc ggg ata cag gtc
cac gag gac tat 192Ser Cys Pro Phe Ser Arg Asn Pro Gly Ile Gln Val
His Glu Asp Tyr 50 55 60 gac tgt acc ctg aac cag acc aac atc ggc
aac aac aac aac aag ttc 240Asp Cys Thr Leu Asn Gln Thr Asn Ile Gly
Asn Asn Asn Asn Lys Phe 65 70 75 80 tat att atc caa ctg ctg gag gag
ggt agt cgc ttc ttc tgc tgg aat 288Tyr Ile Ile Gln Leu Leu Glu Glu
Gly Ser Arg Phe Phe Cys Trp Asn 85 90 95 cgc tgg ggc cgc gtg gga
gag gtg ggc cag agc aag atg aac cac ttc 336Arg Trp Gly Arg Val Gly
Glu Val Gly Gln Ser Lys Met Asn His Phe 100 105 110 acc tgc ctg gaa
gat gca aag aag gac ttt aag aag aaa ttt tgg gag 384Thr Cys Leu Glu
Asp Ala Lys Lys Asp Phe Lys Lys Lys Phe Trp Glu 115 120 125 aag act
aaa aac aaa tgg gag gag cgg gac cgt ttt gtg gcc cag ccc 432Lys Thr
Lys Asn Lys Trp Glu Glu Arg Asp Arg Phe Val Ala Gln Pro 130 135 140
aac aag tac aca ctt ata gaa gtc cag gga gaa gca gag agc caa gag
480Asn Lys Tyr Thr Leu Ile Glu Val Gln Gly Glu Ala Glu Ser Gln Glu
145 150 155 160 gct gta gtg aag gtg gac agc ggc cct gtg agg acc gtg
gtc aag ccc 528Ala Val Val Lys Val Asp Ser Gly Pro Val Arg Thr Val
Val Lys Pro 165 170 175 tgc tcc cta gac cct gcc acc cag aac ctt atc
acc aac atc ttc agc 576Cys Ser Leu Asp Pro Ala Thr Gln Asn Leu Ile
Thr Asn Ile Phe Ser 180 185 190 aaa gag atg ttc aag aac gca atg acc
ctc atg aac ctg gat gtg aag 624Lys Glu Met Phe Lys Asn Ala Met Thr
Leu Met Asn Leu Asp Val Lys 195 200 205 aag atg ccc ttg gga aag ctg
acc aag cag cag att gcc cgt ggc ttc 672Lys Met Pro Leu Gly Lys Leu
Thr Lys Gln Gln Ile Ala Arg Gly Phe 210 215 220 gag gcc ttg gaa gct
cta gag gag gcc atg aaa aac ccc aca ggg gat 720Glu Ala Leu Glu Ala
Leu Glu Glu Ala Met Lys Asn Pro Thr Gly Asp 225 230 235 240 ggc cag
agc ctg gaa gag ctc tcc tcc tgc ttc tac act gtc atc cca 768Gly Gln
Ser Leu Glu Glu Leu Ser Ser Cys Phe Tyr Thr Val Ile Pro 245 250 255
cac aac ttc ggc cgc agc cga ccc ccg ccc atc aac tcc cct gat gtg
816His Asn Phe Gly Arg Ser Arg Pro Pro Pro Ile Asn Ser Pro Asp Val
260 265 270 ctt cag gcc aag aag gac atg ctg ctg gtg cta gcg gac atc
gag ttg 864Leu Gln Ala Lys Lys Asp Met Leu Leu Val Leu Ala Asp Ile
Glu Leu 275 280 285 gcg cag acc ttg cag gca gcc cct ggg gag gag gag
gag aaa gtg gaa 912Ala Gln Thr Leu Gln Ala Ala Pro Gly Glu Glu Glu
Glu Lys Val Glu 290 295 300 gag gtg cca cac cca ctg gat cga gac tac
cag ctc ctc agg tgc cag 960Glu Val Pro His Pro Leu Asp Arg Asp Tyr
Gln Leu Leu Arg Cys Gln 305 310 315 320 ctt caa ctg ctg gac tcc ggg
gag tcc gag tac aag gca ata cag acc 1008Leu Gln Leu Leu Asp Ser Gly
Glu Ser Glu Tyr Lys Ala Ile Gln Thr 325 330 335 tac ctg aaa cag act
ggc aac agc tac agg tgc cca aac ctg cgg cat 1056Tyr Leu Lys Gln Thr
Gly Asn Ser Tyr Arg Cys Pro Asn Leu Arg His 340 345 350 gtt tgg aaa
gtg aac cga gaa ggg gag gga gac agg ttc cag gcc cac 1104Val Trp Lys
Val Asn Arg Glu Gly Glu Gly Asp Arg Phe Gln Ala His 355 360 365 tcc
aaa ctg ggc aat cgg agg ctg ctg tgg cac ggc acc aat gtg gcc
1152Ser Lys Leu Gly Asn Arg Arg Leu Leu Trp His Gly Thr Asn Val Ala
370 375 380 gtg gtg gct gcc atc ctc acc agt ggg ctc cga atc atg cca
cac tcg 1200Val Val Ala Ala Ile Leu Thr Ser Gly Leu Arg Ile Met Pro
His Ser 385 390 395 400 ggt ggt cgt gtt ggc aag ggt att tat ttt gcc
tct gag aac agc aag 1248Gly Gly Arg Val Gly Lys Gly Ile Tyr Phe Ala
Ser Glu Asn Ser Lys 405 410 415 tca gct ggc tat gtt acc acc atg cac
tgt ggg ggc cac cag gtg ggc 1296Ser Ala Gly Tyr Val Thr Thr Met His
Cys Gly Gly His Gln Val Gly 420 425 430 tac atg ttc ctg ggc gag gtg
gcc ctc ggc aaa gag cac cac atc acc 1344Tyr Met Phe Leu Gly Glu Val
Ala Leu Gly Lys Glu His His Ile Thr 435 440 445 atc gat gac ccc agc
ttg aag agt cca ccc cct ggc ttt gac agc gtc 1392Ile Asp Asp Pro Ser
Leu Lys Ser Pro Pro Pro Gly Phe Asp Ser Val 450 455 460 atc gcc cga
ggc caa acc gag ccg gat ccc gcc cag gac att gaa ctt 1440Ile Ala Arg
Gly Gln Thr Glu Pro Asp Pro Ala Gln Asp Ile Glu Leu 465 470 475 480
gaa ctg gat ggg cag ccg gtg gtg gtg ccc caa ggc ccg cct gtg cag
1488Glu Leu Asp Gly Gln Pro Val Val Val Pro Gln Gly Pro Pro Val Gln
485 490 495 tgc ccg tca ttc aaa agc tcc agc ttc agc cag agt gaa tac
ctc ata 1536Cys Pro Ser Phe Lys Ser Ser Ser Phe Ser Gln Ser Glu Tyr
Leu Ile 500 505 510 tac aag gag agc cag tgt cgc ctg cgc tac ctg ctg
gag att cac ctc 1584Tyr Lys Glu Ser Gln Cys Arg Leu Arg Tyr Leu Leu
Glu Ile His Leu 515 520 525 taa 158710528PRTMus musculus 10Met Ala
Pro Lys Arg Lys Ala Ser Val Gln Thr Glu Gly Ser Lys Lys 1 5 10 15
Gln Arg Gln Gly Thr Glu Glu Glu Asp Ser Phe Arg Ser Thr Ala Glu 20
25 30 Ala Leu Arg Ala Ala Pro Ala Asp Asn Arg Val Ile Arg Val Asp
Pro 35 40 45 Ser Cys Pro Phe Ser Arg Asn Pro Gly Ile Gln Val His
Glu Asp Tyr 50 55 60 Asp Cys Thr Leu Asn Gln Thr Asn Ile Gly Asn
Asn Asn Asn Lys Phe 65 70 75 80 Tyr Ile Ile Gln Leu Leu Glu Glu Gly
Ser Arg Phe Phe Cys Trp Asn 85 90 95 Arg Trp Gly Arg Val Gly Glu
Val Gly Gln Ser Lys Met Asn His Phe 100 105 110 Thr Cys Leu Glu Asp
Ala Lys Lys Asp Phe Lys Lys Lys Phe Trp Glu 115 120 125 Lys Thr Lys
Asn Lys Trp Glu Glu Arg Asp Arg Phe Val Ala Gln Pro 130 135 140 Asn
Lys Tyr Thr Leu Ile Glu Val Gln Gly Glu Ala Glu Ser Gln Glu 145 150
155 160 Ala Val Val Lys Val Asp Ser Gly Pro Val Arg Thr Val Val Lys
Pro 165 170 175 Cys Ser Leu Asp Pro Ala Thr Gln Asn Leu Ile Thr Asn
Ile Phe Ser 180 185 190 Lys Glu Met Phe Lys Asn Ala Met Thr Leu Met
Asn Leu Asp Val Lys 195 200 205 Lys Met Pro Leu Gly Lys Leu Thr Lys
Gln Gln Ile Ala Arg Gly Phe 210 215 220 Glu Ala Leu Glu Ala Leu Glu
Glu Ala Met Lys Asn Pro Thr Gly Asp 225 230 235 240 Gly Gln Ser Leu
Glu Glu Leu Ser Ser Cys Phe Tyr Thr Val Ile Pro 245 250 255 His Asn
Phe Gly Arg Ser Arg Pro Pro Pro Ile Asn Ser Pro Asp Val 260 265 270
Leu Gln Ala Lys Lys Asp Met Leu Leu Val Leu Ala Asp Ile Glu Leu 275
280 285 Ala Gln Thr Leu Gln Ala Ala Pro Gly Glu Glu Glu Glu Lys Val
Glu 290 295 300 Glu Val Pro His Pro Leu Asp Arg Asp Tyr Gln Leu Leu
Arg Cys Gln 305 310 315 320 Leu Gln Leu Leu Asp Ser Gly Glu Ser Glu
Tyr Lys Ala Ile Gln Thr 325 330 335 Tyr Leu Lys Gln Thr Gly Asn Ser
Tyr Arg Cys Pro Asn Leu Arg His 340 345 350 Val Trp Lys Val Asn Arg
Glu Gly Glu Gly Asp Arg Phe Gln Ala His 355 360 365 Ser Lys Leu Gly
Asn Arg Arg Leu Leu Trp His Gly Thr Asn Val Ala 370 375 380 Val Val
Ala Ala Ile Leu Thr Ser Gly Leu Arg Ile Met Pro His Ser 385 390 395
400 Gly Gly Arg Val Gly Lys Gly Ile Tyr Phe Ala Ser Glu Asn Ser Lys
405 410 415 Ser Ala Gly Tyr Val Thr Thr Met His Cys Gly Gly His Gln
Val Gly 420 425 430 Tyr Met Phe Leu Gly Glu Val Ala Leu Gly Lys Glu
His His Ile Thr 435 440 445 Ile Asp Asp Pro Ser Leu Lys Ser Pro Pro
Pro Gly Phe Asp Ser Val 450 455 460 Ile Ala Arg Gly Gln Thr Glu Pro
Asp Pro Ala Gln Asp Ile Glu Leu 465 470 475 480 Glu Leu Asp Gly Gln
Pro Val Val Val Pro Gln Gly Pro Pro Val Gln 485 490 495 Cys Pro Ser
Phe Lys Ser Ser Ser Phe Ser Gln Ser Glu Tyr Leu Ile 500 505 510 Tyr
Lys Glu Ser Gln Cys Arg Leu Arg Tyr Leu Leu Glu Ile His Leu 515 520
525 1118PRTArtificial SequenceNAD+ binding domain 11Pro Xaa Xaa Xaa
Xaa Xaa Xaa Gly Xaa Xaa Xaa Gly Lys Gly Ile Tyr 1 5 10 15 Phe Ala
1225PRTArtificial SequenceNAD+ binding domain 12Xaa Xaa Gly Leu Arg
Xaa Xaa Pro Xaa Xaa Xaa Xaa Xaa Xaa Gly Xaa 1 5 10 15 Xaa Xaa Gly
Lys Gly Ile Tyr Phe Ala 20 25 1349PRTArtificial SequenceNAD+
binding domain 13Leu Leu Trp His Gly Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Ile Leu Xaa 1 5 10 15 Xaa Gly Leu Arg Xaa Xaa Pro Xaa Xaa Xaa
Xaa Xaa Xaa Gly Xaa Xaa 20 25 30 Xaa Gly Lys Gly Ile Tyr Phe Ala
Xaa Xaa Xaa Ser Lys Ser Ala Xaa 35 40 45 Tyr 1422PRTArtificial
Sequenceleucine zipper motif 14Xaa Xaa Xaa Xaa Xaa Xaa Xaa Leu Xaa
Xaa Xaa Xaa Xaa Xaa Leu Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Leu 20
1537PRTArtificial Sequencepart-sequence motif 1 15Leu Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Asn Xaa Xaa Tyr Xaa Xaa 1 5 10 15 Gln Leu
Leu Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30
Trp Gly Arg Val Gly 35 1629PRTArtificial Sequencepart-sequence
motif 2 16Ala Xaa Xaa Xaa Phe Xaa Lys Xaa Xaa Xaa Xaa Lys Thr Xaa
Asn Xaa 1 5 10 15 Trp Xaa Xaa Xaa Xaa Xaa Phe Xaa Xaa Xaa Pro Xaa
Lys 20 25 1744PRTArtificial Sequencepart-sequence motif 3 17Gln Xaa
Leu Xaa Xaa Xaa Ile Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15
Met Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Pro Leu Gly Lys Leu 20
25 30 Xaa Xaa Xaa Gln Ile Xaa Xaa Xaa Xaa Xaa Xaa Leu 35 40
1815PRTArtificial Sequencepart-sequence motif 4 18Phe Tyr Thr Xaa
Ile Pro His Xaa Phe Gly Xaa Xaa Xaa Pro Pro 1 5 10 15
1917PRTArtificial Sequencepart-sequence motif 5 19Lys Xaa Xaa Xaa
Leu Xaa Xaa Leu Xaa Asp Ile Glu Xaa Ala Xaa Xaa 1 5 10 15 Leu
2011PRTArtificial Sequencepart-sequence motif 6 20Gly Xaa Xaa Xaa
Leu Xaa Glu Val Ala Leu Gly 1 5 10 2128PRTArtificial
Sequencepart-sequence motif 7 21Gly Xaa Xaa Ser Xaa Xaa Xaa Xaa Gly
Xaa Xaa Xaa Pro Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Leu
Xaa Gly Xaa Xaa Val 20 25 2216PRTArtificial Sequencepart-sequence
motif 8 22Glu Xaa Xaa Xaa Tyr Xaa Xaa Xaa Gln Xaa Xaa Xaa Xaa Tyr
Leu Leu 1 5 10 15 2320PRTArtificial Sequencesynthetic sequence for
antibody production 23Met Ala Ala Arg Arg Arg Arg Ser Thr Gly Gly
Gly Arg Ala Arg Ala 1 5 10 15 Leu Asn Glu Ser 20 2420PRTArtificial
Sequencesynthetic sequence for antibody production 24Lys Thr Glu
Leu Gln Ser Pro Glu His Pro Leu Asp Gln His Tyr Arg 1 5 10 15 Asn
Leu His Cys 20 2521PRTArtificial Sequencesynthetic sequence for
antibody production 25Cys Lys Gly Arg Gln Ala Gly Arg Glu Glu Asp
Pro Phe Arg Ser Thr 1 5 10 15 Ala Glu Ala Leu Lys 20
2620PRTArtificial Sequencesynthetic sequence for antibody
production 26Cys Lys Gln Gln Ile Ala Arg Gly Phe Glu Ala Leu Glu
Ala Leu Glu 1 5 10 15 Glu Ala Leu Lys 20 2719PRTArtificial
Sequencesynthetic sequence for antibody production 27Lys Gln Gln
Ile Ala Arg Gly Phe Glu Ala Leu Glu Ala Leu Glu Glu 1 5 10 15 Ala
Leu Lys 2819PRTMus musculus 28Lys Gln Gln Ile Ala Arg Gly Phe Glu
Ala Leu Glu Ala Leu Glu Glu 1 5 10 15 Ala Met Lys 297PRTArtificial
SequenceNAD+ binding domain 29Gly Xaa Xaa Xaa Gly Lys Gly 1 5
3038PRTArtificial SequencePARP zinc finger sequence motif 30Cys Xaa
Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20
25 30 Xaa Xaa His Xaa Xaa Cys 35 3110PRTArabidopsis thaliana 31Ala
Ala Val Leu Asp Gln Trp Ile Pro Asp 1 5 10 3239DNAHomo
sapiensCDS(1)..(39) 32gta tgc cag gaa ggt cat ggg cca gca aaa ggg
tct ctg 39Gly Met Pro Gly Arg Ser Trp Ala Ser Lys Arg Val Ser 1 5
10 3313PRTHomo sapiens 33Gly Met Pro Gly Arg Ser Trp Ala Ser Lys
Arg Val Ser 1 5 10 34577PRTArtificial Sequencesequence is
hypothetical majority consensus sequence 34Met Ala Gly Gly Leu Arg
Pro Glu Arg Cys Glu Lys Gly Lys Arg Asp 1 5 10 15 Lys Asp Lys Leu
Leu Lys Val Phe Ala Glu Cys Tyr Cys Gly Ala Pro 20 25 30 Lys Arg
Lys Xaa Trp Val Gln Thr Glu Gly Ser Glu Lys Lys Lys Xaa 35 40 45
Arg Gln Xaa Xaa Xaa Glu Glu Asp Xaa Phe Arg Ser Thr Ala Glu Ala 50
55 60 Leu Lys Ala Xaa Pro Ala Glu Xaa Arg Xaa Ile Arg Val Asp Pro
Xaa 65 70 75 80 Cys Pro Leu Ser Xaa Asn Pro Gly Xaa Gln Val Xaa Glu
Asp Val Tyr 85 90 95 Asp Cys Thr Leu Asn Gln Thr Asn Ile Xaa Asn
Asn Asn Asn Lys Phe 100 105 110 Tyr Ile Ile Gln Leu Leu Glu Asp Asp
Xaa Arg Phe Phe Xaa Cys Trp 115 120 125 Asn Arg Trp Gly Arg Val Gly
Glu Val Gly Gln Ser Lys Leu Asn His 130 135 140 Phe Thr Xaa Leu Glu
Asp Ala Lys Glu Asp Phe Xaa Lys Lys Phe Xaa 145 150 155 160 Glu Lys
Glu Thr Lys Asn Asn Trp Glu Glu Arg Asp Xaa Phe Val Lys 165 170 175
Xaa Pro Gly Lys Tyr Thr Leu Leu Glu Val Asp Tyr Xaa Glu Xaa Glu 180
185 190 Asp Glu Glu Ala Val Val Lys Ser Leu Xaa Val Asp Xaa Gly Pro
Val 195 200 205 Ser Thr Val Xaa Lys Arg Val Gln Pro Cys Ser Leu Asp
Pro Ala Thr 210 215 220 Gln Xaa Leu Ile Thr Asn Ile Phe Ser Val Glu
Met Phe Lys Asn Ala 225 230 235 240 Met Xaa Leu Met Xaa Leu Asp Val
Lys Lys Met Pro Leu Gly Lys Leu 245 250 255 Ser Lys Gln Gln Ile Ala
Ala Gly Phe Glu Ala Leu Glu Ala Leu Glu 260 265 270 Glu Ala Xaa Lys
Xaa Gly Thr Xaa Gly Gly Gln Ser Leu Glu Glu Leu 275 280 285 Ser Ser
Xaa Phe Tyr Thr Val Ile Pro His Asp Phe Gly Xaa Ser Xaa 290 295 300
Pro Pro Leu Ile Asn Ser Pro Asp Xaa Leu Gln Ala Lys Lys Asp Met 305
310 315 320 Leu Leu Val Leu Ala Asp Ile Glu Leu Ala Gln Xaa Leu Gln
Ala Xaa 325 330 335 Xaa Xaa Glu Xaa Ser Xaa Lys Val Glu Glu Val Pro
His Pro Leu Asp 340 345 350 Arg Asp Tyr Gln Leu Leu Lys Cys Gln Leu
Gln Leu Leu Asp Ser Gly 355 360 365 Ser Xaa Glu Tyr Lys Val Ile Gln
Thr Tyr Leu Lys Gln Thr Gly Ala 370 375 380 Xaa Thr His Cys Pro Tyr
Thr Leu Xaa Asp Ile Phe Lys Val Glu Arg 385 390 395 400 Glu Gly Glu
Xaa Asp Arg Phe Gln Ala His Ser Lys Leu Gly Asn Arg 405 410 415 Arg
Leu Leu Trp His Gly Ser Asn Met Ala Val Val Ala Gly Ile Leu 420 425
430 Ser Ser Gly Leu Arg Ile Ala Pro His Glu Ala Pro Ser Gly Gly Arg
435 440 445 Val Gly Lys Gly Ile Tyr Phe Ala Ser Glu Asn Ser Lys Ser
Ala Gly 450 455 460 Tyr Val Xaa Thr Ser Xaa Cys Gly Gly His Xaa Val
Gly Leu Met Leu 465 470 475 480 Leu Gly Glu Val Ala Leu Gly Glu His
Glu Leu Xaa Xaa Ala Asn Pro 485 490 495 Ser Leu Lys Ser Leu Pro Pro
Gly Lys Asp Ser Val Ile Gly Leu Gly 500 505 510 Lys Thr Glu Pro Asp
Pro Ala Gln Asp Ile Glu Leu Glu Leu Asp Gly 515 520 525 Gln Gly Val
Val Val Pro Leu Gly Pro Pro Val Xaa Cys Gly Xaa Phe 530 535 540 Xaa
Ser Ser Phe Ser Leu Tyr Ser Glu Tyr Leu Val Tyr Xaa Glu Ser 545 550
555 560 Gln Val Arg Leu Arg Tyr Leu Leu Glu Val His Phe Asn Phe Xaa
Leu 565 570 575 Trp 351014PRTHomo sapiens 35Met Ala Glu Ser Ser Asp
Lys Leu Tyr Arg Val Glu Tyr Ala Lys Ser 1 5 10 15 Glu Arg Ala Ser
Cys Lys Lys Cys Ser Glu Ser Ile Pro Lys Asp Ser 20 25 30 Leu Arg
Met Ala Ile Met Val Gln Ser Pro Met Phe Asp Gly Lys Val 35 40 45
Pro His Trp Tyr His Phe Ser Cys Phe Trp Lys Val Gly His Ser Ile 50
55 60 Arg His Pro Asp Val Glu Val Asp Gly Phe Ser Glu Leu Arg Trp
Asp 65 70 75 80 Asp Gln Gln Lys Val Lys Lys Thr Ala Glu Ala Gly Gly
Val Thr Gly 85 90 95 Lys Gly Gln Asp Gly Ile Gly Ser Lys Ala Glu
Lys Thr Leu Gly Asp 100 105 110 Phe Ala Ala Glu Tyr Ala Lys Ser Asn
Arg Ser Thr Cys Lys Gly Cys 115 120 125 Met Glu Lys Ile Glu Lys Gly
Gln Val Arg Leu Ser Lys Lys Met Val 130 135 140 Asp Pro Glu Lys Pro
Gln Leu Gly Met Ile Asp Arg Trp Tyr His Pro 145 150 155 160 Gly Cys
Phe Val Lys Asn Arg Glu Glu Leu Gly Phe Arg Pro Glu Tyr 165 170 175
Ser Ala Ser Gln Leu Lys Gly Phe Ser Leu Leu Ala Thr Glu Asp Lys 180
185 190 Glu Ala Leu Lys Lys Gln Leu Pro Gly Val Lys Ser Glu Gly Lys
Arg 195 200 205 Lys Gly Asp Lys Val Asp Gly Val Asp Glu Val Ala Lys
Lys Lys Ser 210 215 220 Lys Lys Glu Lys Asp Lys
Asp Ser Lys Leu Glu Lys Ala Leu Lys Ala 225 230 235 240 Gln Asn Asp
Leu Ile Trp Asn Ile Lys Asp Glu Leu Lys Lys Val Cys 245 250 255 Ser
Thr Asn Asp Leu Lys Glu Leu Leu Ile Phe Asn Lys Gln Gln Val 260 265
270 Pro Ser Gly Glu Ser Ala Ile Leu Asp Arg Val Ala Asp Gly Met Val
275 280 285 Phe Gly Ala Leu Leu Pro Cys Glu Glu Cys Ser Gly Gln Leu
Val Phe 290 295 300 Lys Ser Asp Ala Tyr Tyr Cys Thr Gly Asp Val Thr
Ala Trp Thr Lys 305 310 315 320 Cys Met Val Lys Thr Gln Thr Pro Asn
Arg Lys Glu Trp Val Thr Pro 325 330 335 Lys Glu Phe Arg Glu Ile Ser
Tyr Leu Lys Lys Leu Lys Val Lys Lys 340 345 350 Gln Asp Arg Ile Phe
Pro Pro Glu Thr Ser Ala Ser Val Ala Ala Thr 355 360 365 Pro Pro Pro
Ser Thr Ala Ser Ala Pro Ala Ala Val Asn Ser Ser Ala 370 375 380 Ser
Ala Asp Lys Pro Leu Ser Asn Met Lys Ile Leu Thr Leu Gly Lys 385 390
395 400 Leu Ser Arg Asn Lys Asp Glu Val Lys Ala Met Ile Glu Lys Leu
Gly 405 410 415 Gly Lys Leu Thr Gly Thr Ala Asn Lys Ala Ser Leu Cys
Ile Ser Thr 420 425 430 Lys Lys Glu Val Glu Lys Met Asn Lys Lys Met
Glu Glu Val Lys Glu 435 440 445 Ala Asn Ile Arg Val Val Ser Glu Asp
Phe Leu Gln Asp Tyr Ser Ala 450 455 460 Ser Thr Lys Ser Leu Gln Glu
Leu Phe Leu Ala His Ile Leu Ser Pro 465 470 475 480 Trp Gly Ala Glu
Val Lys Ala Glu Pro Val Glu Val Val Ala Pro Arg 485 490 495 Gly Lys
Ser Gly Ala Ala Leu Ser Lys Lys Ser Lys Gly Gln Val Lys 500 505 510
Glu Glu Gly Ile Asn Lys Ser Glu Lys Arg Met Lys Leu Thr Leu Lys 515
520 525 Gly Gly Ala Ala Val Asp Pro Asp Ser Gly Leu Glu His Ser Ala
His 530 535 540 Val Leu Glu Lys Gly Gly Lys Val Phe Ser Ala Thr Leu
Gly Leu Val 545 550 555 560 Asp Ile Val Lys Gly Thr Asn Ser Tyr Tyr
Lys Leu Gln Leu Leu Glu 565 570 575 Asp Asp Lys Glu Asn Arg Tyr Trp
Ile Phe Arg Ser Trp Gly Arg Val 580 585 590 Gly Thr Val Ile Gly Ser
Asn Lys Leu Glu Gln Met Pro Ser Lys Glu 595 600 605 Asp Ala Ile Glu
His Pro Met Lys Leu Tyr Glu Glu Lys Thr Gly Asn 610 615 620 Ala Trp
His Ser Lys Asn Phe Thr Lys Tyr Pro Lys Lys Pro Tyr Pro 625 630 635
640 Leu Glu Ile Asp Tyr Gly Gln Asp Glu Glu Ala Val Lys Lys Leu Thr
645 650 655 Val Asn Pro Gly Thr Lys Ser Lys Leu Pro Lys Pro Val Gln
Asp Leu 660 665 670 Ile Lys Met Ile Pro Asp Val Glu Ser Met Lys Lys
Ala Met Val Glu 675 680 685 Tyr Glu Ile Asp Leu Gln Lys Met Pro Leu
Gly Lys Leu Ser Lys Arg 690 695 700 Gln Ile Gln Ala Ala Tyr Ser Ile
Leu Ser Glu Val Gln Gln Ala Val 705 710 715 720 Ser Gln Gly Ser Ser
Asp Ser Gln Ile Leu Asp Leu Ser Asn Arg Phe 725 730 735 Tyr Thr Leu
Ile Pro His Asp Phe Gly Met Lys Lys Pro Pro Leu Leu 740 745 750 Asn
Asn Ala Asp Ser Val Gln Ala Lys Val Glu Met Leu Asp Asn Leu 755 760
765 Leu Asp Ile Glu Val Ala Tyr Ser Leu Leu Arg Gly Gly Ser Asp Asp
770 775 780 Ser Ser Lys Asp Pro Ile Asp Val Asn Tyr Glu Lys Leu Lys
Thr Asp 785 790 795 800 Ile Lys Val Val Asp Arg Asp Ser Glu Glu Ala
Glu Ile Ile Arg Lys 805 810 815 Tyr Val Lys Asn Thr His Ala Thr Thr
His Asn Ala Tyr Asp Leu Glu 820 825 830 Val Ile Asp Ile Phe Lys Ile
Glu Arg Glu Gly Glu Cys Gln Arg Tyr 835 840 845 Lys Pro Pro Lys Gln
Leu His Asn Arg Arg Leu Leu Trp His Gly Ser 850 855 860 Arg Thr Thr
Asn Phe Ala Gly Ile Leu Ser Gln Gly Leu Arg Ile Ala 865 870 875 880
Pro Pro Glu Ala Pro Val Thr Gly Tyr Met Phe Gly Lys Gly Ile Tyr 885
890 895 Phe Ala Asp Met Val Ser Lys Ser Ala Asn Tyr Cys His Thr Ser
Gln 900 905 910 Gly Asp Pro Ile Gly Leu Ile Leu Leu Gly Glu Val Ala
Leu Gly Asn 915 920 925 Met Tyr Glu Leu Lys His Ala Ser His Ile Ser
Lys Leu Pro Lys Gly 930 935 940 Lys His Ser Val Lys Gly Leu Gly Lys
Thr Thr Pro Asp Pro Ser Ala 945 950 955 960 Asn Ile Ser Leu Asp Gly
Val Asp Val Pro Leu Gly Thr Gly Ile Ser 965 970 975 Ser Gly Val Asn
Asp Thr Ser Leu Leu Tyr Asn Glu Tyr Ile Val Tyr 980 985 990 Asp Ile
Ala Gln Val Asn Leu Lys Tyr Leu Leu Lys Leu Lys Phe Asn 995 1000
1005 Phe Lys Thr Ser Leu Trp 1010 36579PRTArtificial
SequenceMajority sequence from the alignment of human and murine
PARPs 36Met Ala Gly Gly Leu Arg Pro Glu Arg Cys Glu Lys Gly Lys Arg
Asp 1 5 10 15 Lys Asp Lys Leu Leu Lys Val Phe Ala Glu Cys Tyr Cys
Gly Ala Pro 20 25 30 Lys Arg Lys Xaa Trp Val Gln Thr Glu Gly Ser
Glu Lys Lys Lys Xaa 35 40 45 Arg Gln Xaa Xaa Xaa Glu Glu Asp Xaa
Phe Arg Ser Thr Ala Glu Ala 50 55 60 Leu Lys Ala Xaa Pro Ala Glu
Xaa Arg Xaa Ile Arg Val Asp Pro Xaa 65 70 75 80 Cys Pro Leu Ser Xaa
Asn Pro Gly Xaa Gln Val Xaa Glu Asp Val Tyr 85 90 95 Asp Cys Thr
Leu Asn Gln Thr Asn Ile Xaa Asn Asn Asn Asn Lys Phe 100 105 110 Tyr
Ile Ile Gln Leu Leu Glu Asp Asp Xaa Arg Phe Phe Xaa Cys Trp 115 120
125 Asn Arg Trp Gly Arg Val Gly Glu Val Gly Gln Ser Lys Leu Asn His
130 135 140 Phe Thr Xaa Leu Glu Asp Ala Lys Glu Asp Phe Xaa Lys Lys
Phe Xaa 145 150 155 160 Glu Lys Thr Lys Asn Asn Trp Glu Glu Arg Asp
Xaa Phe Val Lys Xaa 165 170 175 Pro Gly Lys Tyr Thr Leu Leu Glu Val
Asp Tyr Xaa Glu Xaa Glu Asp 180 185 190 Glu Glu Ala Val Val Lys Ser
Leu Xaa Val Asp Xaa Gly Pro Val Ser 195 200 205 Thr Val Xaa Lys Arg
Val Gln Pro Cys Ser Leu Asp Pro Ala Thr Gln 210 215 220 Xaa Leu Ile
Thr Asn Ile Phe Ser Val Glu Met Phe Lys Asn Ala Met 225 230 235 240
Xaa Leu Met Xaa Leu Asp Val Lys Lys Met Pro Leu Gly Lys Leu Ser 245
250 255 Lys Gln Gln Ile Ala Ala Gly Phe Glu Ala Leu Glu Ala Leu Glu
Glu 260 265 270 Ala Xaa Lys Xaa Gly Thr Xaa Gly Gly Gln Ser Leu Glu
Glu Leu Ser 275 280 285 Ser Xaa Phe Tyr Thr Val Ile Pro His Asp Phe
Gly Xaa Ser Xaa Pro 290 295 300 Pro Leu Ile Asn Ser Pro Asp Xaa Leu
Gln Ala Lys Lys Asp Met Leu 305 310 315 320 Leu Val Leu Ala Asp Ile
Glu Leu Ala Gln Xaa Leu Gln Ala Xaa Xaa 325 330 335 Xaa Glu Xaa Ser
Xaa Lys Val Glu Glu Val Pro His Pro Leu Asp Arg 340 345 350 Asp Tyr
Gln Leu Leu Lys Cys Gln Leu Gln Leu Leu Asp Ser Gly Ser 355 360 365
Xaa Glu Tyr Lys Val Ile Gln Thr Tyr Leu Lys Gln Thr Gly Ala Xaa 370
375 380 Thr His Cys Pro Tyr Thr Leu Xaa Asp Ile Phe Lys Val Glu Arg
Glu 385 390 395 400 Gly Glu Xaa Asp Arg Phe Gln Ala His Ser Lys Leu
Gly Asn Arg Arg 405 410 415 Leu Leu Trp His Gly Ser Asn Met Ala Val
Val Ala Gly Ile Leu Ser 420 425 430 Ser Gly Leu Arg Ile Ala Pro His
Glu Ala Pro Ser Gly Gly Arg Val 435 440 445 Gly Lys Gly Ile Tyr Phe
Ala Ser Glu Asn Ser Lys Ser Ala Gly Tyr 450 455 460 Val Xaa Thr Ser
Xaa Cys Gly Gly His Xaa Val Gly Leu Met Leu Leu 465 470 475 480 Gly
Glu Val Ala Leu Gly Xaa Glu His Glu Leu Xaa Xaa Ala Asn Pro 485 490
495 Ser Leu Lys Ser Leu Pro Pro Gly Lys Asp Ser Val Ile Gly Leu Gly
500 505 510 Lys Thr Glu Pro Asp Pro Ala Gln Asp Ile Glu Leu Glu Leu
Asp Gly 515 520 525 Gln Gly Val Val Val Pro Leu Gly Pro Pro Val Xaa
Cys Gly Xaa Phe 530 535 540 Xaa Ser Ser Xaa Phe Ser Leu Tyr Ser Glu
Tyr Leu Val Tyr Xaa Glu 545 550 555 560 Ser Gln Val Arg Leu Arg Tyr
Leu Leu Glu Val His Phe Asn Phe Xaa 565 570 575 Xaa Leu Trp
371014PRTHomo sapiens 37Met Ala Glu Ser Ser Asp Lys Leu Tyr Arg Val
Glu Tyr Ala Lys Ser 1 5 10 15 Glu Arg Ala Ser Cys Lys Lys Cys Ser
Glu Ser Ile Pro Lys Asp Ser 20 25 30 Leu Arg Met Ala Ile Met Val
Gln Ser Pro Met Phe Asp Gly Lys Val 35 40 45 Pro His Trp Tyr His
Phe Ser Cys Phe Trp Lys Val Gly His Ser Ile 50 55 60 Arg His Pro
Asp Val Glu Val Asp Gly Phe Ser Glu Leu Arg Trp Asp 65 70 75 80 Asp
Gln Gln Lys Val Lys Lys Thr Ala Glu Ala Gly Gly Val Thr Gly 85 90
95 Lys Gly Gln Asp Gly Ile Gly Ser Lys Ala Glu Lys Thr Leu Gly Asp
100 105 110 Phe Ala Ala Glu Tyr Ala Lys Ser Asn Arg Ser Thr Cys Lys
Gly Cys 115 120 125 Met Glu Lys Ile Glu Lys Gly Gln Val Arg Leu Ser
Lys Lys Met Val 130 135 140 Asp Pro Glu Lys Pro Gln Leu Gly Met Ile
Asp Arg Trp Tyr His Pro 145 150 155 160 Gly Cys Phe Val Lys Asn Arg
Glu Glu Leu Gly Phe Arg Pro Glu Tyr 165 170 175 Ser Ala Ser Gln Leu
Lys Gly Phe Ser Leu Leu Ala Thr Glu Asp Lys 180 185 190 Glu Ala Leu
Lys Lys Gln Leu Pro Gly Val Lys Ser Glu Gly Lys Arg 195 200 205 Lys
Gly Asp Lys Val Asp Gly Val Asp Glu Val Ala Lys Lys Lys Ser 210 215
220 Lys Lys Glu Lys Asp Lys Asp Ser Lys Leu Glu Lys Ala Leu Lys Ala
225 230 235 240 Gln Asn Asp Leu Ile Trp Asn Ile Lys Asp Glu Leu Lys
Lys Val Cys 245 250 255 Ser Thr Asn Asp Leu Lys Glu Leu Leu Ile Phe
Asn Lys Gln Gln Val 260 265 270 Pro Ser Gly Glu Ser Ala Ile Leu Asp
Arg Val Ala Asp Gly Met Val 275 280 285 Phe Gly Ala Leu Leu Pro Cys
Glu Glu Cys Ser Gly Gln Leu Val Phe 290 295 300 Lys Ser Asp Ala Tyr
Tyr Cys Thr Gly Asp Val Thr Ala Trp Thr Lys 305 310 315 320 Cys Met
Val Lys Thr Gln Thr Pro Asn Arg Lys Glu Trp Val Thr Pro 325 330 335
Lys Glu Phe Arg Glu Ile Ser Tyr Leu Lys Lys Leu Lys Val Lys Lys 340
345 350 Gln Asp Arg Ile Phe Pro Pro Glu Thr Ser Ala Ser Val Ala Ala
Thr 355 360 365 Pro Pro Pro Ser Thr Ala Ser Ala Pro Ala Ala Val Asn
Ser Ser Ala 370 375 380 Ser Ala Asp Lys Pro Leu Ser Asn Met Lys Ile
Leu Thr Leu Gly Lys 385 390 395 400 Leu Ser Arg Asn Lys Asp Glu Val
Lys Ala Met Ile Glu Lys Leu Gly 405 410 415 Gly Lys Leu Thr Gly Thr
Ala Asn Lys Ala Ser Leu Cys Ile Ser Thr 420 425 430 Lys Lys Glu Val
Glu Lys Met Asn Lys Lys Met Glu Glu Val Lys Glu 435 440 445 Ala Asn
Ile Arg Val Val Ser Glu Asp Phe Leu Gln Asp Val Ser Ala 450 455 460
Ser Thr Lys Ser Leu Gln Glu Leu Phe Leu Ala His Ile Leu Ser Pro 465
470 475 480 Trp Gly Ala Glu Val Lys Ala Glu Pro Val Glu Val Val Ala
Pro Arg 485 490 495 Gly Lys Ser Gly Ala Ala Leu Ser Lys Lys Ser Lys
Gly Gln Val Lys 500 505 510 Glu Glu Gly Ile Asn Lys Ser Glu Lys Arg
Met Lys Leu Thr Leu Lys 515 520 525 Gly Gly Ala Ala Val Asp Pro Asp
Ser Gly Leu Glu His Ser Ala His 530 535 540 Val Leu Glu Lys Gly Gly
Lys Val Phe Ser Ala Thr Leu Gly Leu Val 545 550 555 560 Asp Ile Val
Lys Gly Thr Asn Ser Tyr Tyr Lys Leu Gln Leu Leu Glu 565 570 575 Asp
Asp Lys Glu Asn Arg Tyr Trp Ile Phe Arg Ser Trp Gly Arg Val 580 585
590 Gly Thr Val Ile Gly Ser Asn Lys Leu Glu Gln Met Pro Ser Lys Glu
595 600 605 Asp Ala Ile Glu His Phe Met Lys Leu Tyr Glu Glu Lys Thr
Gly Asn 610 615 620 Ala Trp His Ser Lys Asn Phe Thr Lys Tyr Pro Lys
Lys Phe Tyr Pro 625 630 635 640 Leu Glu Ile Asp Tyr Gly Gln Asp Glu
Glu Ala Val Lys Lys Leu Thr 645 650 655 Val Asn Pro Gly Thr Lys Ser
Lys Leu Pro Lys Pro Val Gln Asp Leu 660 665 670 Ile Lys Met Ile Phe
Asp Val Glu Ser Met Lys Lys Ala Met Val Glu 675 680 685 Tyr Glu Ile
Asp Leu Gln Lys Met Pro Leu Gly Lys Leu Ser Lys Arg 690 695 700 Gln
Ile Gln Ala Ala Tyr Ser Ile Leu Ser Glu Val Gln Gln Ala Val 705 710
715 720 Ser Gln Gly Ser Ser Asp Ser Gln Ile Leu Asp Leu Ser Asn Arg
Phe 725 730 735 Tyr Thr Leu Ile Pro His Asp Phe Gly Met Lys Lys Pro
Pro Leu Leu 740 745 750 Asn Asn Ala Asp Ser Val Gln Ala Lys Val Glu
Met Leu Asp Asn Leu 755 760 765 Leu Asp Ile Glu Val Ala Tyr Ser Leu
Leu Arg Gly Gly Ser Asp Asp 770 775 780 Ser Ser Lys Asp Pro Ile Asp
Val Asn Tyr Glu Lys Leu Lys Thr Asp 785 790 795 800 Ile Lys Val Val
Asp Arg Asp Ser Glu Glu Ala Glu Ile Ile Arg Lys 805 810 815 Tyr Val
Lys Asn Thr His Ala Thr Thr His Asn Ala Tyr Asp Leu Glu 820 825 830
Val Ile Asp Ile Phe Lys Ile Glu Arg Glu Gly Glu Cys Gln Arg Tyr 835
840 845 Lys Pro Phe Lys Gln Leu His Asn Arg Arg Leu Leu Trp His Gly
Ser 850 855 860 Arg Thr Thr Asn Phe Ala Gly Ile Leu Ser Gln Gly Leu
Arg Ile Ala 865 870 875 880 Pro Pro Glu Ala Pro Val Thr Gly Tyr Met
Phe Gly Lys Gly Ile Tyr 885 890 895 Phe Ala Asp Met Val Ser Lys Ser
Ala Asn Tyr Cys His Thr Ser Gln 900 905 910 Gly Asp Pro Ile Gly Leu
Ile Leu Leu Gly Glu Val Ala Leu Gly Asn 915 920
925 Met Tyr Glu Leu Lys His Ala Ser His Ile Ser Lys Leu Pro Lys Gly
930 935 940 Lys His Ser Val Lys Gly Leu Gly Lys Thr Thr Pro Asp Pro
Ser Ala 945 950 955 960 Asn Ile Ser Leu Asp Gly Val Asp Val Pro Leu
Gly Thr Gly Ile Ser 965 970 975 Ser Gly Val Asn Asp Thr Ser Leu Leu
Tyr Asn Glu Tyr Ile Val Tyr 980 985 990 Asp Ile Ala Gln Val Asn Leu
Lys Tyr Leu Leu Lys Leu Lys Phe Asn 995 1000 1005 Phe Lys Thr Ser
Leu Trp 1010 38570PRTHomo sapiens 38Met Ala Ala Arg Arg Arg Arg Ser
Thr Gly Gly Gly Arg Ala Arg Ala 1 5 10 15 Leu Asn Glu Ser Lys Arg
Val Asn Asn Gly Asn Thr Ala Pro Glu Asp 20 25 30 Ser Ser Pro Ala
Lys Lys Thr Arg Arg Cys Gln Arg Gln Glu Ser Lys 35 40 45 Lys Met
Pro Val Ala Gly Gly Lys Ala Asn Lys Asp Arg Thr Glu Asp 50 55 60
Lys Gln Asp Glu Ser Val Lys Ala Leu Leu Leu Lys Gly Lys Ala Pro 65
70 75 80 Val Asp Pro Glu Cys Thr Ala Lys Val Gly Lys Ala His Val
Tyr Cys 85 90 95 Glu Gly Asn Asp Val Tyr Asp Val Met Leu Asn Gln
Thr Asn Leu Gln 100 105 110 Phe Asn Asn Asn Lys Tyr Tyr Leu Ile Gln
Leu Leu Glu Asp Asp Ala 115 120 125 Gln Arg Asn Phe Ser Val Trp Met
Arg Trp Gly Arg Val Gly Lys Met 130 135 140 Gly Gln His Ser Leu Val
Ala Cys Ser Gly Asn Leu Asn Lys Ala Lys 145 150 155 160 Glu Ile Phe
Gln Lys Lys Phe Leu Asp Lys Thr Lys Asn Asn Trp Glu 165 170 175 Asp
Arg Glu Lys Phe Glu Lys Val Pro Gly Lys Tyr Asp Met Leu Gln 180 185
190 Met Asp Tyr Ala Thr Asn Thr Gln Asp Glu Glu Glu Thr Lys Lys Glu
195 200 205 Glu Ser Leu Lys Ser Pro Leu Lys Pro Glu Ser Gln Leu Asp
Leu Arg 210 215 220 Val Gln Glu Leu Ile Lys Leu Ile Cys Asn Val Gln
Ala Met Glu Glu 225 230 235 240 Met Met Met Glu Met Lys Tyr Asn Thr
Lys Lys Ala Pro Leu Gly Lys 245 250 255 Leu Thr Val Ala Gln Ile Lys
Ala Gly Tyr Gln Ser Leu Lys Lys Ile 260 265 270 Glu Asp Cys Ile Arg
Ala Gly Gln His Gly Arg Ala Leu Met Glu Ala 275 280 285 Cys Asn Glu
Phe Tyr Thr Arg Ile Pro His Asp Phe Gly Leu Arg Thr 290 295 300 Pro
Pro Leu Ile Arg Thr Gln Lys Glu Leu Ser Glu Lys Ile Gln Leu 305 310
315 320 Leu Glu Ala Leu Gly Asp Ile Glu Ile Ala Ile Lys Leu Val Lys
Thr 325 330 335 Glu Leu Gln Ser Pro Glu His Pro Leu Asp Gln His Tyr
Arg Asn Leu 340 345 350 His Cys Ala Leu Arg Pro Leu Asp His Glu Ser
Tyr Glu Phe Lys Val 355 360 365 Ile Ser Gln Tyr Leu Gln Ser Thr His
Ala Pro Thr His Ser Asp Tyr 370 375 380 Thr Met Thr Leu Leu Asp Leu
Phe Glu Val Glu Lys Asp Gly Glu Lys 385 390 395 400 Glu Ala Phe Arg
Glu Asp Leu His Asn Arg Met Leu Leu Trp His Gly 405 410 415 Ser Arg
Met Ser Asn Trp Val Gly Ile Leu Ser His Gly Leu Arg Ile 420 425 430
Ala Pro Pro Glu Ala Pro Ile Thr Gly Tyr Met Phe Gly Lys Gly Ile 435
440 445 Tyr Phe Ala Asp Met Ser Ser Lys Ser Ala Asn Tyr Cys Phe Ala
Ser 450 455 460 Arg Leu Lys Asn Thr Gly Leu Leu Leu Leu Ser Glu Val
Ala Leu Gly 465 470 475 480 Gln Cys Asn Glu Leu Leu Glu Ala Asn Pro
Lys Ala Glu Gly Leu Leu 485 490 495 Gln Gly Lys His Ser Thr Lys Gly
Leu Gly Lys Met Ala Pro Ser Ser 500 505 510 Ala His Phe Val Thr Leu
Asn Gly Ser Thr Val Pro Leu Gly Pro Ala 515 520 525 Ser Asp Thr Gly
Ile Leu Asn Pro Asp Gly Tyr Thr Leu Asn Tyr Asn 530 535 540 Glu Tyr
Ile Val Tyr Asn Pro Asn Gln Val Arg Met Arg Tyr Leu Leu 545 550 555
560 Lys Val Gln Phe Asn Phe Leu Gln Leu Trp 565 57039540PRTHomo
sapiens 39Met Ser Leu Leu Phe Leu Ala Met Ala Pro Lys Pro Lys Pro
Trp Val 1 5 10 15 Gln Thr Glu Gly Pro Glu Lys Lys Lys Gly Arg Gln
Ala Gly Arg Glu 20 25 30 Glu Asp Pro Phe Arg Ser Thr Ala Glu Ala
Leu Lys Ala Ile Pro Ala 35 40 45 Glu Lys Arg Ile Ile Arg Val Asp
Pro Thr Cys Pro Leu Ser Ser Asn 50 55 60 Pro Gly Thr Gln Val Tyr
Glu Asp Tyr Asn Cys Thr Leu Asn Gln Thr 65 70 75 80 Asn Ile Glu Asn
Asn Asn Asn Lys Phe Tyr Ile Ile Gln Leu Leu Gln 85 90 95 Asp Ser
Asn Arg Phe Phe Thr Cys Trp Asn Arg Trp Gly Arg Val Gly 100 105 110
Glu Val Gly Gln Ser Lys Ile Asn His Phe Thr Arg Leu Glu Asp Ala 115
120 125 Lys Lys Asp Phe Glu Lys Lys Phe Arg Glu Lys Thr Lys Asn Asn
Trp 130 135 140 Ala Glu Arg Asp His Phe Val Ser His Pro Gly Lys Tyr
Thr Leu Ile 145 150 155 160 Glu Val Gln Ala Glu Asp Glu Ala Gln Glu
Ala Val Val Lys Val Asp 165 170 175 Arg Gly Pro Val Arg Thr Val Thr
Lys Arg Val Gln Pro Cys Ser Leu 180 185 190 Asp Pro Ala Thr Gln Lys
Leu Ile Thr Asn Ile Phe Ser Lys Glu Met 195 200 205 Phe Lys Asn Thr
Met Ala Leu Met Asp Leu Asp Val Lys Lys Met Pro 210 215 220 Leu Gly
Lys Leu Ser Lys Gln Gln Ile Ala Arg Gly Phe Glu Ala Leu 225 230 235
240 Glu Ala Leu Glu Glu Ala Leu Lys Gly Pro Thr Asp Gly Gly Gln Ser
245 250 255 Leu Glu Glu Leu Ser Ser His Phe Tyr Thr Val Ile Pro His
Asn Phe 260 265 270 Gly His Ser Gln Pro Pro Pro Ile Asn Ser Pro Glu
Leu Leu Gln Ala 275 280 285 Lys Lys Asp Met Leu Leu Val Leu Ala Asp
Ile Glu Leu Ala Gln Ala 290 295 300 Leu Gln Ala Val Ser Glu Gln Glu
Lys Thr Val Glu Glu Val Pro His 305 310 315 320 Pro Leu Asp Arg Asp
Tyr Gln Leu Leu Lys Cys Gln Leu Gln Leu Leu 325 330 335 Asp Ser Gly
Ala Pro Glu Tyr Lys Val Ile Gln Thr Tyr Leu Glu Gln 340 345 350 Thr
Gly Ser Asn His Arg Cys Pro Thr Leu Gln His Ile Trp Lys Val 355 360
365 Asn Gln Glu Gly Glu Glu Asp Arg Phe Gln Ala His Ser Lys Leu Gly
370 375 380 Asn Arg Lys Leu Leu Trp His Gly Thr Asn Met Ala Val Val
Ala Ala 385 390 395 400 Ile Leu Thr Ser Gly Leu Arg Ile Met Pro His
Ser Gly Gly Arg Val 405 410 415 Gly Lys Gly Ile Tyr Phe Ala Ser Glu
Asn Ser Lys Ser Ala Gly Tyr 420 425 430 Val Ile Gly Met Lys Cys Gly
Ala His His Val Gly Tyr Met Phe Leu 435 440 445 Gly Glu Val Ala Leu
Gly Arg Glu His His Ile Asn Thr Asp Asn Pro 450 455 460 Ser Leu Lys
Ser Pro Pro Pro Gly Phe Asp Ser Val Ile Ala Arg Gly 465 470 475 480
His Thr Glu Pro Asp Pro Thr Gln Asp Thr Glu Leu Glu Leu Asp Gly 485
490 495 Gln Gln Val Val Val Pro Gln Gly Gln Pro Val Pro Cys Pro Glu
Phe 500 505 510 Ser Ser Ser Thr Phe Ser Gln Ser Glu Tyr Leu Ile Tyr
Gln Glu Ser 515 520 525 Gln Cys Arg Leu Arg Tyr Leu Leu Glu Val His
Leu 530 535 540 40533PRTMus musculus 40Met Ala Pro Lys Arg Lys Ala
Ser Val Gln Thr Glu Gly Ser Lys Lys 1 5 10 15 Gln Arg Gln Gly Thr
Glu Glu Glu Asp Ser Phe Arg Ser Thr Ala Glu 20 25 30 Ala Leu Arg
Ala Ala Pro Ala Asp Asn Arg Val Ile Arg Val Asp Pro 35 40 45 Ser
Cys Pro Phe Ser Arg Asn Pro Gly Ile Gln Val His Glu Asp Tyr 50 55
60 Asp Cys Thr Leu Asn Gln Thr Asn Ile Gly Asn Asn Asn Asn Lys Phe
65 70 75 80 Tyr Ile Ile Gln Leu Leu Glu Glu Gly Ser Arg Phe Phe Cys
Trp Asn 85 90 95 Arg Trp Gly Arg Val Gly Glu Val Gly Gln Ser Lys
Met Asn His Phe 100 105 110 Thr Cys Leu Glu Asp Ala Lys Lys Asp Phe
Lys Lys Lys Phe Trp Glu 115 120 125 Lys Thr Lys Asn Lys Trp Glu Glu
Arg Asp Arg Phe Val Ala Gln Pro 130 135 140 Asn Lys Tyr Thr Leu Ile
Glu Val Gln Gly Glu Ala Glu Ser Gln Glu 145 150 155 160 Ala Val Val
Lys Ala Leu Ser Pro Gln Val Asp Ser Gly Pro Val Arg 165 170 175 Thr
Val Val Lys Pro Cys Ser Leu Asp Pro Ala Thr Gln Asn Leu Ile 180 185
190 Thr Asn Ile Phe Ser Lys Glu Met Phe Lys Asn Ala Met Thr Leu Met
195 200 205 Asn Leu Asp Val Lys Lys Met Pro Leu Gly Lys Leu Thr Lys
Gln Gln 210 215 220 Ile Ala Arg Gly Phe Glu Ala Leu Glu Ala Leu Glu
Glu Ala Met Lys 225 230 235 240 Asn Pro Thr Gly Asp Gly Gln Ser Leu
Glu Glu Leu Ser Ser Cys Phe 245 250 255 Tyr Thr Val Ile Pro His Asn
Phe Gly Arg Ser Arg Pro Pro Pro Ile 260 265 270 Asn Ser Pro Asp Val
Leu Gln Ala Lys Lys Asp Met Leu Leu Val Leu 275 280 285 Ala Asp Ile
Glu Leu Ala Gln Thr Leu Gln Ala Ala Pro Gly Glu Glu 290 295 300 Glu
Glu Lys Val Glu Glu Val Pro His Pro Leu Asp Arg Asp Tyr Gln 305 310
315 320 Leu Leu Arg Cys Gln Leu Gln Leu Leu Asp Ser Gly Glu Ser Glu
Tyr 325 330 335 Lys Ala Ile Gln Thr Tyr Leu Lys Gln Thr Gly Asn Ser
Tyr Arg Cys 340 345 350 Pro Asn Leu Arg His Val Trp Lys Val Asn Arg
Glu Gly Glu Gly Asp 355 360 365 Arg Phe Gln Ala His Ser Lys Leu Gly
Asn Arg Arg Leu Leu Trp His 370 375 380 Gly Thr Asn Val Ala Val Val
Ala Ala Ile Leu Thr Ser Gly Leu Arg 385 390 395 400 Ile Met Pro His
Ser Gly Gly Arg Val Gly Lys Gly Ile Tyr Phe Ala 405 410 415 Ser Glu
Asn Ser Lys Ser Ala Gly Tyr Val Thr Thr Met His Cys Gly 420 425 430
Gly His Gln Val Gly Tyr Met Phe Leu Gly Glu Val Ala Leu Gly Lys 435
440 445 Glu His His Ile Thr Ile Asp Asp Pro Ser Leu Lys Ser Pro Pro
Pro 450 455 460 Gly Phe Asp Ser Val Ile Ala Arg Gly Gln Thr Glu Pro
Asp Pro Ala 465 470 475 480 Gln Asp Ile Glu Leu Glu Leu Asp Gly Gln
Pro Val Val Val Pro Gln 485 490 495 Gly Pro Pro Val Gln Cys Pro Ser
Phe Lys Ser Ser Ser Phe Ser Gln 500 505 510 Ser Glu Tyr Leu Ile Tyr
Lys Glu Ser Gln Cys Arg Leu Arg Tyr Leu 515 520 525 Leu Glu Ile His
Leu 530
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